Adam6 mice

ABSTRACT

Mice are provided that comprise a reduction or deletion of ADAM6 activity from an endogenous ADAM6 locus, or that lack an endogenous locus encoding a mouse ADAM6 protein, wherein the mice comprise a sequence encoding an ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the sequence is an ectopic ADAM6 sequence or a sequence that confers upon a male mouse the ability to generate offspring by mating. Mice and cells with genetically modified immunoglobulin heavy chain loci that comprise an ectopic nucleotide sequence encoding a mouse ADAM6 or functional fragment or homolog or ortholog thereof are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC §119(e) of U.S.Provisional Application Ser. No. 61/595,200, filed Feb. 6, 2012, U.S.Provisional Application Ser. No. 61/497,650, filed Jun. 16, 2011 andU.S. Provisional Application Ser. No. 61/446,895, filed Feb. 25, 2011,which applications are hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

Genetically modified mice, cells, embryos, and tissues that comprise anucleic acid sequence encoding a functional ADAM6 locus are described.Modifications include human and/or humanized immunoglobulin loci. Micethat lack a functional endogenous ADAM6 gene but that comprise ADAM6function are described, including mice that comprise an ectopic nucleicacid sequence that encodes an ADAM6 protein. Genetically modified malemice that comprise a modification of an endogenous immunoglobulin V_(H)locus that renders the mouse incapable of making a functional ADAM6protein and results in a loss in fertility, and that further compriseADAM6 function in the male mice are described, including mice thatcomprise an ectopic nucleic acid sequence that restores fertility to themale mouse.

BACKGROUND OF INVENTION

Mice that contain human antibody genes are known in the art.Pharmaceutical applications for antibodies in the last two decades havefueled a great deal of research into making antibodies that are suitablefor use as human therapeutics. Early antibody therapeutics, which werebased on mouse antibodies, were not ideal as human therapeutics becauserepeated administration of mouse antibodies to humans results inimmunogenicity that can confound long-term treatment regimens. Solutionsbased on humanizing mouse antibodies to make them appear more human andless mouse-like were developed. Methods for expressing humanimmunoglobulin sequences for use in antibodies followed, mostly based onin vitro expression of human immunoglobulin libraries in phage,bacteria, or yeast. Finally, attempts were made to make useful humanantibodies from human lymphocytes in vitro, in mice engrafted with humanhematopoietic cells, and in transchromosomal or transgenic mice withdisabled endogenous immunoglobulin loci. In the transgenic mice, it wasnecessary to disable the endogenous mouse immunoglobulin genes so thatthe randomly integrated fully human transgenes would function as thesource of immunoglobulin sequences expressed in the mouse. Such mice canmake human antibodies suitable for use as human therapeutics, but thesemice display substantial problems with their immune systems. Theseproblems (1) make the mice impractical for generating a sufficientlydiverse antibody repertoire, (2) require the use of extensivere-engineering fixes, (3) provide a suboptimal clonal selection processlikely due to incompatibility between human and mouse elements, and (4)render these mice an unreliable source of large and diverse populationsof human variable sequences needed to be truly useful for making humantherapeutics.

There remains a need in the art for making improved genetically modifiedmice that are useful in generating immunoglobulin sequences, includinghuman antibody sequences. There also remains a need for mice that arecapable of rearranging immunoglobulin gene segments to form usefulrearranged immunoglobulin genes, or capable of making proteins fromaltered immunoglobulin loci, while at the same time reducing oreliminating deleterious changes that might result from the geneticmodifications.

SUMMARY OF INVENTION

In one aspect, nucleic acid constructs, cells, embryos, mice, andmethods are provided for making mice that comprise a modification thatresults in a nonfunctional endogenous mouse ADAM6 protein or ADAM6 gene(e.g., a knockout of or a deletion in an endogenous ADAM6 gene), whereinthe mice comprise a nucleic acid sequence that encodes an ADAM6 proteinor ortholog or homolog or fragment thereof that is functional in a malemouse.

In one aspect, nucleic acid constructs, cells, embryos, mice, andmethods are provided for making mice that comprise a modification of anendogenous mouse immunoglobulin locus, wherein the mice comprise anADAM6 protein or ortholog or homolog or fragment thereof that isfunctional in a male mouse. In one embodiment, the endogenous mouseimmunoglobulin locus is an immunoglobulin heavy chain locus, and themodification reduces or eliminates ADAM6 activity of a cell or tissue ofa male mouse.

In one aspect, mice are provided that comprise an ectopic nucleotidesequence encoding a mouse ADAM6 or ortholog or homolog or functionalfragment thereof; mice are also provided that comprise an endogenousnucleotide sequence encoding a mouse ADAM6 or ortholog or homolog orfragment thereof, and at least one genetic modification of a heavy chainimmunoglobulin locus.

In one aspect, methods are provided for making mice that comprise amodification of an endogenous mouse immunoglobulin locus, wherein themice comprise an ADAM6 protein or ortholog or homolog or fragmentthereof that is functional in a male mouse.

In one aspect, methods are provided for making mice that comprise agenetic modification of an immunoglobulin heavy chain locus, whereinapplication of the methods result in male mice that comprise a modifiedimmunoglobulin heavy chain locus (or a deletion thereof), and the malemice are capable of generating offspring by mating. In one embodiment,the male mice are capable of producing sperm that can transit from amouse uterus through a mouse oviduct to fertilize a mouse egg.

In one aspect, methods are provided for making mice that comprise agenetic modification of an immunoglobulin heavy chain locus, whereinapplication of the methods result in male mice that comprise a modifiedimmunoglobulin heavy chain locus (or a deletion thereof), and the malemice exhibit a reduction in fertility, and the mice comprise a geneticmodification that restores in whole or in part the reduction infertility. In various embodiments, the reduction in fertility ischaracterized by an inability of the sperm of the male mice to migratefrom a mouse uterus through a mouse oviduct to fertilize a mouse egg. Invarious embodiments, the reduction in fertility is characterized bysperm that exhibit an in vivo migration defect. In various embodiments,the genetic modification that restores in whole or in part the reductionin fertility is a nucleic acid sequence encoding a mouse ADAM6 gene orortholog or homolog or fragment thereof that is functional in a malemouse.

In one embodiment, the genetic modification comprises replacingendogenous immunoglobulin heavy chain variable loci with immunoglobulinheavy chain variable loci of another species (e.g., a non-mousespecies). In one embodiment, the genetic modification comprisesinsertion of orthologous immunoglobulin heavy chain variable loci intoendogenous immunoglobulin heavy chain variable loci. In a specificembodiment, the species is human. In one embodiment, the geneticmodification comprises deletion of an endogenous immunoglobulin heavychain variable locus in whole or in part, wherein the deletion resultsin a loss of endogenous ADAM6 function. In a specific embodiment, theloss of endogenous ADAM6 function is associated with a reduction infertility in male mice.

In one aspect, mice are provided that comprise a modification thatreduces or eliminates mouse ADAM6 expression from an endogenous ADAM6allele such that a male mouse having the modification exhibits a reducedfertility (e.g., a highly reduced ability to generate offspring bymating), or is essentially infertile, due to the reduction orelimination of endogenous ADAM6 function, wherein the mice furthercomprise an ectopic ADAM6 sequence or homolog or ortholog or functionalfragment thereof. In one aspect, the modification that reduces oreliminates mouse ADAM6 expression is a modification (e.g., an insertion,a deletion, a replacement, etc.) in a mouse immunoglobulin locus.

In one embodiment, the reduction or loss of ADAM6 function comprises aninability or substantial inability of the mouse to produce sperm thatcan travel from a mouse uterus through a mouse oviduct to fertilize amouse egg. In a specific embodiment, at least about 95%, 96%, 97%, 98%,or 99% of the sperm cells produced in an ejaculate volume of the mouseare incapable of traversing through an oviduct in vivo followingcopulation and fertilizing a mouse ovum.

In one embodiment, the reduction or loss of ADAM6 function comprises aninability to form or substantial inability to form a complex of ADAM2and/or ADAM3 and/or ADAM6 on a surface of a sperm cell of the mouse. Inone embodiment, the loss of ADAM6 function comprises a substantialinability to fertilize a mouse egg by copulation with a female mouse.

In one aspect, a mouse is provided that lacks a functional endogenousADAM6 gene, and comprises a protein (or an ectopic nucleotide sequencethat encodes a protein) that confers ADAM6 functionality on the mouse.In one embodiment, the mouse is a male mouse and the functionalitycomprises enhanced fertility as compared with a mouse that lacks afunctional endogenous ADAM6 gene.

In one embodiment, the protein is encoded by a genomic sequence locatedwithin an immunoglobulin locus in the germline of the mouse. In aspecific embodiment, the immunoglobulin locus is a heavy chain locus. Inanother specific embodiment, the heavy chain locus comprises at leastone human V_(H), at least one human D_(H) and at least one human J_(H)gene segment. In one embodiment, the ectopic protein is encoded by agenomic sequence located within a non-immunoglobulin locus in thegermline of the mouse. In one embodiment, the non-immunoglobulin locusis a transcriptionally active locus. In a specific embodiment, thetranscriptionally active locus is the ROSA26 locus. In a specificembodiment, the transcriptionally active locus is associated withtissue-specific expression. In one embodiment, the tissue-specificexpression is present in reproductive tissues. In one embodiment, theprotein is encoded by a genomic sequence randomly inserted into thegermline of the mouse.

In one embodiment, the mouse comprises a human or chimeric human/mouseor chimeric human/rat light chain (e.g., human variable, mouse or ratconstant) and a chimeric human variable/mouse or rat constant heavychain. In a specific embodiment, the mouse comprises a transgene thatcomprises a chimeric human variable/rat or mouse constant light chaingene operably linked to a transcriptionally active promoter, e.g., aROSA26 promoter. In a further specific embodiment, the chimerichuman/mouse or rat light chain transgene comprises a rearranged humanlight chain variable region sequence in the germline of the mouse.

In one embodiment, the ectopic nucleotide sequence is located within animmunoglobulin locus in the germline of the mouse. In a specificembodiment, the immunoglobulin locus is a heavy chain locus. In oneembodiment, the heavy chain locus comprises at least one human V_(H), atleast one human D_(H) and at least one human J_(H) gene segment. In oneembodiment, the ectopic nucleotide sequence is located within anon-immunoglobulin locus in the germline of the mouse. In oneembodiment, the non-immunoglobulin locus is a transcriptionally activelocus. In a specific embodiment, the transcriptionally active locus isthe ROSA26 locus. In one embodiment, the ectopic nucleotide sequence ispositioned randomly inserted into the germline of the mouse.

In one aspect, a mouse is provided that lacks a functional endogenousADAM6 gene, wherein the mouse comprises an ectopic nucleotide sequencethat complements the loss of mouse ADAM6 function. In one embodiment,the ectopic nucleotide sequence confers upon the mouse an ability toproduce offspring that is comparable to a corresponding wild-type mousethat contains a functional endogenous ADAM6 gene. In one embodiment, thesequence confers upon the mouse an ability to form a complex of ADAM2and/or ADAM3 and/or ADAM6 on the surface of sperm cell of the mouse. Inone embodiment, the sequence confers upon the mouse an ability to travelfrom a mouse uterus through a mouse oviduct to a mouse ovum to fertilizethe ovum.

In one embodiment, the mouse lacking the functional endogenous ADAM6gene and comprising the ectopic nucleotide sequence produces at leastabout 50%, 60%, 70%, 80%, or 90% of the number of litters a wild-typemouse of the same age and strain produces in a six-month time period.

In one embodiment, the mouse lacking the functional endogenous ADAM6gene and comprising the ectopic nucleotide sequence produces at leastabout 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about4-fold, about 6-fold, about 7-fold, about 8-fold, or about 10-fold ormore progeny when bred over a six-month time period than a mouse of thesame age and the same or similar strain that lacks the functionalendogenous ADAM6 gene and that lacks the ectopic nucleotide sequencethat is bred over substantially the same time period and undersubstantially the same conditions.

In one embodiment, the mouse lacking the functional endogenous ADAM6gene and comprising the ectopic nucleotide sequence produces an averageof at least about 2-fold, 3-fold, or 4-fold higher number of pups perlitter in a 4- or 6-month breeding period than a mouse that lacks thefunctional endogenous ADAM6 gene and that lacks the ectopic nucleotidesequence, and that is bred for the same period of time.

In one embodiment, the mouse lacking the functional endogenous ADAM6gene and comprising the ectopic nucleotide sequence is a male mouse, andthe male mouse produces sperm that when recovered from oviducts at about5-6 hours post-copulation reflects an oviduct migration that is at least10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least90-fold, 100-fold, 110-fold, or 120-fold or higher than a mouse thatlacks the functional endogenous ADAM6 gene and that lacks the ectopicnucleotide sequence.

In one embodiment, the mouse lacking the functional endogenous ADAM6gene and comprising the ectopic nucleotide sequence when copulated witha female mouse generates sperm that is capable of traversing the uterusand entering and traversing the oviduct within about 6 hours at anefficiency that is about equal to sperm from a wild-type mouse.

In one embodiment, the mouse lacking the functional endogenous ADAM6gene and comprising the ectopic nucleotide sequence produces about1.5-fold, about 2-fold, about 3-fold, or about 4-fold or more litters ina comparable period of time than a mouse that lacks the functional ADAM6gene and that lacks the ectopic nucleotide sequence.

In one aspect, a mouse comprising in its germline a non-mouse nucleicacid sequence that encodes an immunoglobulin protein is provided,wherein the non-mouse immunoglobulin sequence comprises an insertion ofa mouse ADAM6 gene or homolog or ortholog or functional fragmentthereof. In one embodiment, the non-mouse immunoglobulin sequencecomprises a human immunoglobulin sequence. In one embodiment, thesequence comprises a human immunoglobulin heavy chain sequence. In oneembodiment, the sequence comprises a human immunoglobulin light chainsequence. In one embodiment, the sequence comprises one or more V genesegments, one or more D gene segments, and one or more J gene segments;in one embodiment, the sequence comprises one or more V gene segmentsand one or more J gene segments. In one embodiment, the one or more V,D, and J gene segments, or one or more V and J gene segments, are notrearranged. In one embodiment, the one or more V, D, and J genesegments, or one or more V and J gene segments, are rearranged. In oneembodiment, following rearrangement of the one or more V, D, and J genesegments, or one or more V and J gene segments, the mouse comprises inits genome at least one nucleic acid sequence encoding a mouse ADAM6gene or homolog or ortholog or functional fragment thereof. In oneembodiment, following rearrangement the mouse comprises in its genome atleast two nucleic acid sequences encoding a mouse ADAM6 gene or homologor ortholog or functional fragment thereof. In one embodiment, followingrearrangement the mouse comprises in its genome at least one nucleicacid sequence encoding a mouse ADAM6 gene or homolog or ortholog orfunctional fragment thereof. In one embodiment, the mouse comprises theADAM6 gene or homolog or ortholog or functional fragment thereof in a Bcell. In one embodiment, the mouse comprises the ADAM6 gene or homologor ortholog or functional fragment thereof in a non-B cell.

In one aspect, mice are provided that express a human immunoglobulinheavy chain variable region or functional fragment thereof from anendogenous mouse immunoglobulin heavy chain locus, wherein the micecomprise an ADAM6 activity that is functional in a male mouse.

In one embodiment, the male mice comprise a single unmodified endogenousADAM6 allele or ortholog of homolog or functional fragment thereof at anendogenous ADAM6 locus.

In one embodiment, the male mice comprise an ectopic mouse ADAM6sequence or homolog or ortholog or functional fragment thereof thatencodes a protein that confers ADAM6 function.

In one embodiment, the male mice comprise an ADAM6 sequence or homologor ortholog or functional fragment thereof at a location in the mousegenome that approximates the location of the endogenous mouse ADAM6allele, e.g., 3′ of a final V gene segment sequence and 5′ of an initialD gene segment.

In one embodiment, the male mice comprise an ADAM6 sequence or homologor ortholog or functional fragment thereof flanked upstream, downstream,or upstream and downstream (with respect to the direction oftranscription of the ADAM6 sequence) of a nucleic acid sequence encodingan immunoglobulin variable gene segment. In a specific embodiment, theimmunoglobulin variable gene segment is a human gene segment. In oneembodiment, the immunoglobulin variable gene segment is a human genesegment, and the sequence encoding the mouse ADAM6 or ortholog orhomolog or fragment thereof functional in a mouse is between human Vgene segments; in one embodiment, the mouse comprises two or more humanV gene segments, and the sequence is at a position between the final Vgene segment and the penultimate V gene segment; in one embodiment, thesequence is at a position following the final V gene segment and thefirst D gene segment.

In one aspect, a male mouse is provided that comprises a nonfunctionalendogenous ADAM6 gene, or a deletion of an endogenous ADAM6 gene, in itsgermline; wherein sperm cells of the mouse are capable of transiting anoviduct of a female mouse and fertilizing an egg. In one embodiment, themice comprise an extrachromosomal copy of a mouse ADAM6 gene or orthologor homolog or functional fragment thereof that is functional in a malemouse. In one embodiment, the mice comprise an ectopic mouse ADAM6 geneor ortholog or homolog or functional fragment thereof that is functionalin a male mouse.

In one aspect, mice are provided that comprise a genetic modificationthat reduces endogenous mouse ADAM6 function, wherein the mousecomprises at least some ADAM6 functionality provided either by anendogenous unmodified allele that is functional in whole or in part(e.g., a heterozygote), or by expression from an ectopic sequence thatencodes an ADAM6 or an ortholog or homolog or functional fragmentthereof that is functional in a male mouse.

In one embodiment, the mice comprise ADAM6 function sufficient to conferupon male mice the ability to generate offspring by mating, as comparedwith male mice that lack a functional ADAM6. In one embodiment, theADAM6 function is conferred by the presence of an ectopic nucleotidesequence that encodes a mouse ADAM6 or a homolog or ortholog orfunctional fragment thereof. ADAM6 homologs or orthologs or fragmentsthereof that are functional in a male mouse include those that restore,in whole or in part, the loss of ability to generate offspring observedin a male mouse that lacks sufficient endogenous mouse ADAM6 activity,e.g., the loss in ability observed in an ADAM6 knockout mouse. In thissense ADAM6 knockout mice include mice that comprise an endogenous locusor fragment thereof, but that is not functional, i.e., that does notexpress ADAM6 (ADAM6a and/or ADAM6b) at all, or that expresses ADAM6(ADAM6a and/or ADAM6b) at a level that is insufficient to support anessentially normal ability to generate offspring of a wild-type malemouse. The loss of function can be due, e.g., to a modification in astructural gene of the locus (i.e., in an ADAM6a or ADAM6b codingregion) or in a regulatory region of the locus (e.g., in a sequence 5′to the ADAM6a gene, or 3′ of the ADAM6a or ADAM6b coding region, whereinthe sequence controls, in whole or in part, transcription of an ADAM6gene, expression of an ADAM6 RNA, or expression of an ADAM6 protein). Invarious embodiments, orthologs or homologs or fragments thereof that arefunctional in a male mouse are those that enable a sperm of a male mouse(or a majority of sperm cells in the ejaculate of a male mouse) totransit a mouse oviduct and fertilize a mouse ovum.

In one embodiment, male mice that express the human immunoglobulinvariable region or functional fragment thereof comprise sufficient ADAM6activity to confer upon the male mice the ability to generate offspringby mating with female mice and, in one embodiment, the male mice exhibitan ability to generate offspring when mating with female mice that is inone embodiment at least 25%, in one embodiment, at least 30%, in oneembodiment at least 40%, in one embodiment at least 50%, in oneembodiment at least 60%, in one embodiment at least 70%, in oneembodiment at least 80%, in one embodiment at least 90%, and in oneembodiment about the same as, that of mice with one or two endogenousunmodified ADAM6 alleles.

In one embodiment male mice express sufficient ADAM6 (or an ortholog orhomolog or functional fragment thereof) to enable a sperm cell from themale mice to traverse a female mouse oviduct and fertilize a mouse egg.

In one embodiment, the ADAM6 functionality is conferred by a nucleicacid sequence that is contiguous with a mouse chromosomal sequence(e.g., the nucleic acid is randomly integrated into a mouse chromosome;or placed at a specific location, e.g., by targeting the nucleic acid toa specific location, e.g., by site-specific recombinase-mediated (e.g.,Cre-mediated) insertion or homologous recombination). In one embodiment,the ADAM6 sequence is present on a nucleic acid that is distinct from achromosome of the mouse (e.g., the ADAM6 sequence is present on anepisome, i.e., extrachromosomally, e.g., in an expression construct, avector, a YAC, a transchromosome, etc.).

In one aspect, genetically modified mice and cells are provided thatcomprise a modification of an endogenous immunoglobulin heavy chainlocus, wherein the mice express at least a portion of an immunoglobulinheavy chain sequence, e.g., at least a portion of a human sequence,wherein the mice comprise an ADAM6 activity that is functional in a malemouse. In one embodiment, the modification reduces or eradicates ADAM6activity of the mouse. In one embodiment, the mouse is modified suchthat both alleles that encode ADAM6 activity are either absent orexpress an ADAM6 that does not substantially function to support normalmating in a male mouse. In one embodiment, the mouse further comprisesan ectopic nucleic acid sequence encoding a mouse ADAM6 or ortholog orhomolog or functional fragment thereof.

In one aspect, genetically modified mice and cells are provided thatcomprise a modification of an endogenous immunoglobulin heavy chainlocus, wherein the modification reduces or eliminates ADAM6 activityexpressed from an ADAM6 sequence of the locus, and wherein the micecomprise an ADAM6 protein or ortholog or homolog or functional fragmentthereof. In various embodiments, the ADAM6 protein or fragment thereofis encoded by an ectopic ADAM6 sequence. In various embodiments, theADAM6 protein or fragment thereof is expressed from an endogenous ADAM6allele. In various embodiments, the mouse comprises a firstimmunoglobulin heavy chain allele comprises a first modification thatreduces or eliminates expression of a functional ADAM6 from the firstimmunoglobulin heavy chain allele, and the mouse comprises a secondimmunoglobulin heavy chain allele that comprises a second modificationthat does not substantially reduce or does not eliminate expression of afunctional ADAM6 from the second immunoglobulin heavy chain allele.

In one embodiment, the second modification is located 3′ (with respectto the transcriptional directionality of the mouse V gene segment) of afinal mouse V gene segment and located 5′ (with respect to thetranscriptional directionality of the constant sequence) of a mouse (orchimeric human/mouse) immunoglobulin heavy chain constant gene orfragment thereof (e.g., a nucleic acid sequence encoding a human and/ormouse: C_(H)1 and/or hinge and/or C_(H)2 and/or C_(H)3).

In one embodiment, the modification is at a first immunoglobulin heavychain allele at a first locus that encodes a first ADAM6 allele, and theADAM6 function results from expression of an endogenous ADAM6 at asecond immunoglobulin heavy chain allele at a second locus that encodesa functional ADAM6, wherein the second immunoglobulin heavy chain allelecomprises at least one modification of a V, D, and/or J gene segment. Ina specific embodiment, the at least one modification of the V, D, and orJ gene segment is a deletion, a replacement with a human V, D, and/or Jgene segment, a replacement with a camelid V, D, and/or J gene segment,a replacement with a humanized or camelized V, D, and/or J gene segment,a replacement of a heavy chain sequence with a light chain sequence, anda combination thereof. In one embodiment, the at least one modificationis the deletion of one or more heavy chain V, D, and/or J gene segmentsand a replacement with one or more light chain V and/or J gene segments(e.g., a human light chain V and/or J gene segment) at the heavy chainlocus.

In one embodiment, the modification is at a first immunoglobulin heavychain allele at a first locus and a second immunoglobulin heavy chainallele at a second locus, and the ADAM6 function results from expressionof an ectopic ADAM6 at a non-immunoglobulin locus in the germline of themouse. In a specific embodiment, the non-immunoglobulin locus is theROSA26 locus. In a specific embodiment, the non-immunoglobulin locus istranscriptionally active in reproductive tissue.

In one aspect, a mouse comprising a heterozygous or a homozygousknockout of ADAM6 is provided. In one embodiment, the mouse furthercomprises a modified immunoglobulin sequence that is a human or ahumanized immunoglobulin sequence, or a camelid or camelized human ormouse immunoglobulin sequence. In one embodiment, the modifiedimmunoglobulin sequence is present at the endogenous mouse heavy chainimmunoglobulin locus. In one embodiment, the modified immunoglobulinsequence comprises a human heavy chain variable gene sequence at anendogenous mouse immunoglobulin heavy chain locus. In one embodiment,the human heavy chain variable gene sequence replaces an endogenousmouse heavy chain variable gene sequence at the endogenous mouseimmunoglobulin heavy chain locus.

In one aspect, a mouse incapable of expressing a functional endogenousmouse ADAM6 from an endogenous mouse ADAM6 locus is provided. In oneembodiment, the mouse comprises an ectopic nucleic acid sequence thatencodes an ADAM6, or functional fragment thereof, that is functional inthe mouse. In a specific embodiment, the ectopic nucleic acid sequenceencodes a protein that rescues a loss in the ability to generateoffspring exhibited by a male mouse that is homozygous for an ADAM6knockout. In a specific embodiment, the ectopic nucleic acid sequenceencodes a mouse ADAM6 protein.

In one aspect, a mouse is provided that lacks a functional endogenousADAM6 locus, and that comprises an ectopic nucleic acid sequence thatconfers upon the mouse ADAM6 function. In one embodiment, the nucleicacid sequence comprises an endogenous mouse ADAM6 sequence or functionalfragment thereof. In one embodiment, the endogenous mouse ADAM6 sequencecomprises ADAM6a- and ADAM6b-encoding sequence located in a wild-typemouse between the 3′-most mouse immunoglobulin heavy chain V genesegment (V_(H)) and the 5′-most mouse immunoglobulin heavy chain D genesegment (D_(H)).

In one embodiment, the nucleic acid sequence comprises a sequenceencoding mouse ADAM6a or functional fragment thereof and/or a sequenceencoding mouse ADAM6b or functional fragment thereof, wherein the ADAM6aand/or ADAM6b or functional fragment(s) thereof is operably linked to apromoter. In one embodiment, the promoter is a human promoter. In oneembodiment, the promoter is the mouse ADAM6 promoter. In a specificembodiment, the ADAM6 promoter comprises sequence located between thefirst codon of the first ADAM6 gene closest to the mouse 5′-most D_(H)gene segment and the recombination signal sequence of the 5′-most D_(H)gene segment, wherein 5′ is indicated with respect to direction oftranscription of the mouse immunoglobulin genes. In one embodiment, thepromoter is a viral promoter. In a specific embodiment, the viralpromoter is a cytomegalovirus (CMV) promoter. In one embodiment, thepromoter is a ubiquitin promoter.

In one embodiment, the promoter is an inducible promoter. In oneembodiment, the inducible promoter regulates expression innon-reproductive tissues. In one embodiment, the inducible promoterregulates expression in reproductive tissues. In a specific embodiment,the expression of the mouse ADAM6a and/or ADAM6b sequences or functionalfragments(s) thereof is developmentally regulated by the induciblepromoter in reproductive tissues.

In one embodiment, the mouse ADAM6a and/or ADAM6b are selected from theADAM6a of SEQ ID NO:1 and/or ADAM6b of sequence SEQ ID NO:2. In oneembodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO:3. In aspecific embodiment, the mouse ADAM6 promoter comprises the nucleic acidsequence of SEQ ID NO:3 directly upstream (with respect to the directionof transcription of ADAM6a) of the first codon of ADAM6a and extendingto the end of SEQ ID NO:3 upstream of the ADAM6 coding region. Inanother specific embodiment, the ADAM6 promoter is a fragment extendingfrom within about 5 to about 20 nucleotides upstream of the start codonof ADAM6a to about 0.5 kb, 1 kb, 2 kb, or 3 kb or more upstream of thestart codon of ADAM6a.

In one embodiment, the nucleic acid sequence comprises SEQ ID NO:3 or afragment thereof that when placed into a mouse that is infertile or thathas low fertility due to a lack of ADAM6, improves fertility or restoresfertility to about a wild-type fertility. In one embodiment, SEQ ID NO:3or a fragment thereof confers upon a male mouse the ability to produce asperm cell that is capable of traversing a female mouse oviduct in orderto fertilize a mouse egg.

In one aspect, a mouse is provided that comprises a deletion of anendogenous nucleotide sequence that encodes an ADAM6 protein, areplacement of an endogenous mouse V_(H) gene segment with a human V_(H)gene segment, and an ectopic nucleotide sequence that encodes a mouseADAM6 protein or ortholog or homolog or fragment thereof that isfunctional in a male mouse.

In one embodiment, the mouse comprises an immunoglobulin heavy chainlocus that comprises a deletion of an endogenous immunoglobulin locusnucleotide sequence that comprises an endogenous ADAM6 gene, comprises anucleotide sequence encoding one or more human immunoglobulin genesegments, and wherein the ectopic nucleotide sequence encoding the mouseADAM6 protein is within or directly adjacent to the nucleotide sequenceencoding the one or more human immunoglobulin gene segments.

In one embodiment, the mouse comprises a replacement of all orsubstantially all endogenous V_(H) gene segments with a nucleotidesequence encoding one or more human V_(H) gene segments, and the ectopicnucleotide sequence encoding the mouse ADAM6 protein is within, ordirectly adjacent to, the nucleotide sequence encoding the one or morehuman V_(H) gene segments. In one embodiment, the mouse furthercomprises a replacement of one or more endogenous D_(H) gene segmentswith one or more human D_(H) gene segments at the endogenous D_(H) genelocus. In one embodiment, the mouse further comprises a replacement ofone or more endogenous J_(H) gene segments with one or more human J_(H)gene segments at the endogenous J_(H) gene locus. In one embodiment, themouse comprises a replacement of all or substantially all endogenousV_(H), D_(H), and J_(H) gene segments and a replacement at theendogenous V_(H), D_(H), and J_(H) gene loci with human V_(H), D_(H),and J_(H) gene segments, wherein the mouse comprises an ectopic sequenceencoding a mouse ADAM6 protein. In a specific embodiment, the ectopicsequence encoding the mouse ADAM6 protein is placed between thepenultimate 3′-most V_(H) gene segment of the human V_(H) gene segmentspresent, and the ultimate 3′ V_(H) gene segment of the human V_(H) genesegments present. In a specific embodiment, the mouse comprises adeletion of all or substantially all mouse V_(H) gene segments, and areplacement with all or substantially all human V_(H) gene segments, andthe ectopic nucleotide sequence encoding the mouse ADAM6 protein isplaced downstream of human gene segment V_(H)1-2 and upstream of humangene segment V_(H)6-1.

In a specific embodiment, the mouse comprises a replacement of all orsubstantially all endogenous V_(H) gene segments with a nucleotidesequence encoding one or more human V_(H) gene segments, and the ectopicnucleotide sequence encoding the mouse ADAM6 protein is within, ordirectly adjacent to, the nucleotide sequence encoding the one or morehuman V_(H) gene segments.

In one embodiment, the ectopic nucleotide sequence that encodes themouse ADAM6 protein is present on a transgene in the genome of themouse. In one embodiment, the ectopic nucleotide sequence that encodesthe mouse ADAM6 protein is present extrachromosomally in the mouse.

In one aspect, a mouse is provided that comprises a modification of anendogenous immunoglobulin heavy chain locus, wherein the mouse expressesa B cell that comprises a rearranged immunoglobulin sequence operablylinked to a heavy chain constant region gene sequence, and the B cellcomprises in its genome (e.g., on a B cell chromosome) a gene encodingan ADAM6 or ortholog or homolog or fragment thereof that is functionalin a male mouse. In one embodiment, the rearranged immunoglobulinsequence operably linked to the heavy chain constant region genesequence comprises a human heavy chain V, D, and/or J sequence; a mouseheavy chain V, D, and/or J sequence; a human or mouse light chain Vand/or J sequence. In one embodiment, the heavy chain constant regiongene sequence comprises a human or a mouse heavy chain sequence selectedfrom the group consisting of a C_(H)1, a hinge, a C_(H)2, a C_(H)3, anda combination thereof.

In one aspect, a genetically modified mouse is provided, wherein themouse comprises a functionally silenced immunoglobulin light chain gene,and further comprises a replacement of one or more endogenousimmunoglobulin heavy chain variable region gene segments with one ormore human immunoglobulin heavy chain variable region gene segments,wherein the mouse lacks a functional endogenous ADAM6 locus, and whereinthe mouse comprises an ectopic nucleotide sequence that expresses amouse ADAM6 protein or an ortholog or homolog or fragment thereof thatis functional in a male mouse.

In one aspect, a mouse is provided that lacks a functional endogenousmouse ADAM6 locus or sequence and that comprises an ectopic nucleotidesequence encoding a mouse ADAM6 locus or functional fragment of a mouseADAM6 locus or sequence, wherein the mouse is capable of mating with amouse of the opposite sex to produce a progeny that comprises theectopic ADAM6 locus or sequence. In one embodiment, the mouse is male.In one embodiment, the mouse is female.

In one aspect, a genetically modified mouse is provided, wherein themouse comprises a human immunoglobulin heavy chain variable region genesegment at an endogenous mouse immunoglobulin heavy chain variableregion gene locus, the mouse lacks an endogenous functional ADAM6sequence at the endogenous mouse immunoglobulin heavy chain variableregion gene locus, and wherein the mouse comprises an ectopic nucleotidesequence that expresses a mouse ADAM6 protein or an ortholog or homologor fragment thereof that is functional in a male mouse.

In one embodiment, the ectopic nucleotide sequence that expresses themouse ADAM6 protein is extrachromosomal. In one embodiment, the ectopicnucleotide sequence that expresses the mouse ADAM6 protein is integratedat one or more loci in a genome of the mouse. In a specific embodiment,the one or more loci include an immunoglobulin locus.

In one aspect, a mouse is provided that expresses an immunoglobulinheavy chain sequence from a modified endogenous mouse immunoglobulinheavy chain locus, wherein the heavy chain is derived from a human Vgene segment, a D gene segment, and a J gene segment, wherein the mousecomprises an ADAM6 activity that is functional in the mouse.

In one embodiment, the mouse comprises a plurality of human V genesegments, a plurality of D gene segments, and a plurality of J genesegments. In one embodiment, the D gene segments are human D genesegments. In one embodiment, the J gene segments are human J genesegments. In one embodiment, the mouse further comprises a humanizedheavy chain constant region sequence, wherein the humanization comprisesreplacement of a sequence selected from a C_(H)1, hinge, C_(H)2, C_(H)3,and a combination thereof. In a specific embodiment, the heavy chain isderived from a human V gene segment, a human D gene segment, a human Jgene segment, a human C_(H)1 sequence, a human or mouse hinge sequence,a mouse C_(H)2 sequence, and a mouse C_(H)3 sequence. In anotherspecific embodiment, the mouse further comprises a human light chainconstant sequence.

In one embodiment, the D gene segment is flanked 5′ (with respect totranscriptional direction of the D gene segment) by a sequence encodingan ADAM6 activity that is functional in the mouse.

In one embodiment, the ADAM6 activity that is functional in the mouseresults from expression of a nucleotide sequence located 5′ of the5′-most D gene segment and 3′ of the 3′-most V gene segment (withrespect to the direction of transcription of the V gene segment) of themodified endogenous mouse heavy chain immunoglobulin locus.

In one embodiment, the ADAM6 activity that is functional in the mouseresults from expression of a nucleotide sequence located between twohuman V gene segments in the modified endogenous mouse heavy chainimmunoglobulin locus. In one embodiment, the two human V gene segmentsare a human V_(H)1-2 gene segment and a V_(H)6-1 gene segment.

In one embodiment, the nucleotide sequence comprises a sequence selectedfrom a mouse ADAM6b sequence or functional fragment thereof, a mouseADAM6a sequence or functional fragment thereof, and a combinationthereof.

In one embodiment, the nucleotide sequence between the two human V genesegments is placed in opposite transcription orientation with respect tothe human V gene segments. In a specific embodiment, nucleotide sequenceencodes, from 5′ to 3′ with respect to the direction of transcription ofADAM6 genes, and ADAM6a sequence followed by an ADAM6b sequence.

In one embodiment, the mouse comprises a replacement of a human ADAM6pseudogene sequence between human V gene segments V_(H)1-2 and V_(H)6-1with a mouse ADAM6 sequence or a functional fragment thereof.

In one embodiment, the sequence encoding the ADAM6 activity that isfunctional in the mouse is a mouse ADAM6 sequence or functional fragmentthereof.

In one embodiment, the mouse comprises an endogenous mouse DFL16.1 genesegment (e.g., in a mouse heterozygous for the modified endogenous mouseimmunoglobulin heavy chain locus), or a human D_(H)1-1 gene segment. Inone embodiment, the D gene segment of the immunoglobulin heavy chainexpressed by the mouse is derived from an endogenous mouse DFL16.1 genesegment or a human D_(H)1-1 gene segment.

In one aspect, a mouse is provided that comprises a nucleic acidsequence encoding a mouse ADAM6 (or homolog or ortholog or functionalfragment thereof) in a DNA-bearing cell of non-rearranged B celllineage, but does not comprise the nucleic acid sequence encoding themouse ADAM6 (or homolog or ortholog or functional fragment thereof) in aB cell that comprise rearranged immunoglobulin loci, wherein the nucleicacid sequence encoding the mouse ADAM6 (or homolog or ortholog orfunctional fragment thereof) occurs in the genome at a position that isdifferent from a position in which a mouse ADAM6 gene appears in awild-type mouse. In one embodiment, the nucleic acid sequence encodingthe mouse ADAM6 (or homolog or ortholog or functional fragment thereof)is present in all or substantially all DNA-bearing cells that are not ofrearranged B cell lineage; in one embodiment, the nucleic acid sequenceis present in germline cells of the mouse, but not in a chromosome of arearranged B cell.

In one aspect, a mouse is provided that comprises a nucleic acidsequence encoding a mouse ADAM6 (or homolog or ortholog or functionalfragment thereof) in all or substantially all DNA-bearing cells,including B cells that comprise rearranged immunoglobulin loci, whereinthe nucleic acid sequence encoding the mouse ADAM6 (or homolog orortholog or functional fragment thereof) occurs in the genome at aposition that is different from a position in which a mouse ADAM6 geneappears in a wild-type mouse. In one embodiment, the nucleic acidsequence encoding the mouse ADAM6 (or homolog or ortholog or functionalfragment thereof) is on a nucleic acid that is contiguous with therearranged immunoglobulin locus. In one embodiment, the nucleic acidthat is contiguous with the rearranged immunoglobulin locus is achromosome. In one embodiment, the chromosome is a chromosome that isfound in a wild-type mouse and the chromosome comprises a modificationof a mouse immunoglobulin locus.

In one aspect, a genetically modified mouse is provided, wherein themouse comprises a B cell that comprises in its genome an ADAM6 sequenceor ortholog or homolog thereof. In one embodiment, the ADAM6 sequence orortholog or homolog thereof is at an immunoglobulin heavy chain locus.In one embodiment, the ADAM6 sequence or ortholog or homolog thereof isat a locus that is not an immunoglobulin locus. In one embodiment, theADAM6 sequence is on a transgene driven by a heterologous promoter. In aspecific embodiment, the heterologous promoter is a non-immunoglobulinpromoter. In a specific embodiment, B cell expresses an ADAM6 protein orortholog or homolog thereof.

In one embodiment, 90% or more of the B cells of the mouse comprise agene encoding an ADAM6 protein or an ortholog thereof or a homologthereof or a fragment thereof that is functional in the mouse. In aspecific embodiment, the mouse is a male mouse.

In one embodiment, the B cell genome comprises a first allele and asecond allele comprising the ADAM6 sequence or ortholog or homologthereof. In one embodiment, the B cell genome comprises a first allelebut not a second allele comprising the ADAM6 sequence or ortholog orhomolog thereof.

In one aspect, a mouse is provided that comprises a modification at oneor more endogenous ADAM6 alleles.

In one embodiment, the modification renders the mouse incapable ofexpressing a functional ADAM6 protein from at least one of the one ormore endogenous ADAM6 alleles. In a specific embodiment, the mouse isincapable of expressing a functional ADAM6 protein from each of theendogenous ADAM6 alleles.

In one embodiment, the mice are incapable of expressing a functionalADAM6 protein from each endogenous ADAM6 allele, and the mice comprisean ectopic ADAM6 sequence.

In one embodiment, the mice are incapable of expressing a functionalADAM6 protein from each endogenous ADAM6 allele, and the mice comprisean ectopic ADAM6 sequence located within 1, 2, 3, 4, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 110, or 120 or more kb upstream (with respectto the direction of transcription of the mouse heavy chain locus) of amouse immunoglobulin heavy chain constant region sequence. In a specificembodiment, the ectopic ADAM6 sequence is at the endogenousimmunoglobulin heavy chain locus (e.g., in an intergenic V-D region,between two V gene segments, between a V and a D gene segment, between aD and a J gene segment, etc.). In a specific embodiment, the ectopicADAM6 sequence is located within a 90 to 100 kb intergenic sequencebetween the final mouse V gene segment and the first mouse D genesegment. In another specific embodiment, the endogenous 90 to 100 kbintergenic V-D sequence is removed, and the ectopic ADAM6 sequence isplaced between the final V and the first D gene segment.

In one aspect, an infertile male mouse is provided, wherein the mousecomprises a deletion of two or more endogenous ADAM6 alleles. In oneaspect, a female mouse is provided that is a carrier of a maleinfertility trait, wherein the female mouse comprises in its germline anonfunctional ADAM6 allele or a knockout of an endogenous ADAM6 allele.

In one aspect, a mouse that lacks an endogenous immunoglobulin heavychain V, D, and J gene segment is provided, wherein a majority of the Bcells of the mouse comprise an ADAM6 sequence or ortholog or homologthereof.

In one embodiment, the mouse lacks endogenous immunoglobulin heavy chaingene segments selected from two or more V gene segments, two or more Dgene segments, two or more J gene segments, and a combination thereof.In one embodiment, the mouse lacks immunoglobulin heavy chain genesegments selected from at least one and up to 89 V gene segments, atleast one and up to 13 D gene segments, at least one and up to four Jgene segments, and a combination thereof. In one embodiment, the mouselacks a genomic DNA fragment from chromosome 12 comprising about threemegabases of the endogenous immunoglobulin heavy chain locus. In aspecific embodiment, the mouse lacks all functional endogenous heavychain V, D, and J gene segments. In a specific embodiment, the mouselacks 89 V_(H) gene segments, 13 D_(H) gene segments and four J_(H) genesegments.

In one aspect, a mouse is provided, wherein the mouse has a genome inthe germline comprising a modification of an immunoglobulin heavy chainlocus, wherein the modification to the immunoglobulin heavy chain locuscomprises the replacement of one or more mouse immunoglobulin variableregion sequences with one or more non-mouse immunoglobulin variableregion sequences, and wherein the mouse comprises a nucleic acidsequence encoding a mouse ADAM6 protein. In a preferred embodiment, theD_(H) and J_(H) sequences and at least 3, at least 10, at least 20, atleast 40, at least 60, or at least 80 V_(H) sequences of theimmunoglobulin heavy chain locus are replaced by non-mouseimmunoglobulin variable region sequences. In a further preferredembodiment, the D_(H), J_(H), and all V_(H) sequences of theimmunoglobulin heavy chain locus are replaced by non-mouseimmunoglobulin variable region sequences. The non-mouse immunoglobulinvariable region sequences can be non-rearranged. In a preferredembodiment, the non-mouse immunoglobulin variable region sequencescomprise complete non-rearranged D_(H) and J_(H) regions and at least 3,at least 10, at least 20, at least 40, at least 60, or at least 80non-rearranged V_(H) sequences of the non-mouse species. In a furtherpreferred embodiment, the non-mouse immunoglobulin variable regionsequences comprise the complete variable region, including all V_(H),D_(H), and J_(H) regions, of the non-mouse species. The non-mousespecies can be Homo sapiens and the non-mouse immunoglobulin variableregion sequences can be human sequences.

In one aspect, a mouse that expresses an antibody that comprises atleast one human variable domain/non-human constant domain immunoglobulinpolypeptide is provided, wherein the mouse expresses a mouse ADAM6protein or ortholog or homolog thereof from a locus other than animmunoglobulin locus.

In one embodiment, the ADAM6 protein or ortholog or homolog thereof isexpressed in a B cell of the mouse, wherein the B cell comprises arearranged immunoglobulin sequence that comprises a human variablesequence and a non-human constant sequence.

In one embodiment, the non-human constant sequence is a rodent sequence.In one embodiment, the rodent is selected from a mouse, a rat, and ahamster.

In one aspect, a method is provided for making an infertile male mouse,comprising rendering an endogenous ADAM6 allele of a donor ES cellnonfunctional (or knocking out said allele), introducing the donor EScell into a host embryo, gestating the host embryo in a surrogatemother, and allowing the surrogate mother to give birth to progenyderived in whole or in part from the donor ES cell. In one embodiment,the method further comprises breeding progeny to obtain an infertilemale mouse.

In one aspect, a method is provided for making a mouse with a geneticmodification of interest, wherein the mouse is infertile, the methodcomprising the steps of (a) making a genetic modification of interest ina genome; (b) modifying the genome to knockout an endogenous ADAM6allele, or render an endogenous ADAM6 allele nonfunctional; and, (c)employing the genome in making a mouse. In various embodiments, thegenome is from an ES cell or used in a nuclear transfer experiment.

In one aspect, a mouse made using a targeting vector, nucleotideconstruct, or cell as described herein is provided.

In one aspect, a progeny of a mating of a mouse as described herein witha second mouse that is a wild-type mouse or genetically modified isprovided.

In one aspect, a method for maintaining a mouse strain is provided,wherein the mouse strain comprises a replacement of a mouseimmunoglobulin heavy chain sequence with one or more heterologousimmunoglobulin heavy chain sequences. In one embodiment, the one or moreheterologous immunoglobulin heavy chain sequences are humanimmunoglobulin heavy chain sequences.

In one embodiment, the mouse strain comprises a deletion of one or moremouse V_(H), D_(H), and/or J_(H) gene segments. In one embodiment, themouse further comprises one or more human V_(H) gene segments, one ormore human D_(H) gene segments, and/or one or more human J_(H) genesegments. In one embodiment, the mouse comprises at least 3, at least10, at least 20, at least 40, at least 60, or at least 80 human V_(H)segments, at least 27 human D_(H) gene segments, and at least six J_(H)gene segments. In a specific embodiment, the mouse comprises at least 3,at least 10, at least 20, at least 40, at least 60, or at least 80 humanV_(H) segments, the at least 27 human D_(H) gene segments, and the atleast six J_(H) gene segments are operably linked to a constant regiongene. In one embodiment, the constant region gene is a mouse constantregion gene. In one embodiment, the constant region gene comprises amouse constant region gene sequence selected from a C_(H)1, a hinge, aC_(H)2, a C_(H)3, and/or a C_(H)4 or a combination thereof.

In one embodiment, the method comprises generating a male mouseheterozygous for the replacement of the mouse immunoglobulin heavy chainsequence, and breeding the heterozygous male mouse with a wild-typefemale mouse or a female mouse that is homozygous or heterozygous forthe human heavy chain sequence. In one embodiment, the method comprisesmaintaining the strain by repeatedly breeding heterozygous males withfemales that are wild type or homozygous or heterozygous for the humanheavy chain sequence.

In one embodiment, the method comprises obtaining cells from male orfemale mice homozygous or heterozygous for the human heavy chainsequence, and employing those cells as donor cells or nuclei therefromas donor nuclei, and using the cells or nuclei to make geneticallymodified animals using host cells and/or gestating the cells and/ornuclei in surrogate mothers.

In one embodiment, only male mice that are heterozygous for thereplacement at the heavy chain locus are bred to female mice. In aspecific embodiment, the female mice are homozygous, heterozygous, orwild type with respect to a replaced heavy chain locus.

In one embodiment, the mouse further comprises a replacement of λ and/orκ light chain variable sequences at an endogenous immunoglobulin lightchain locus with heterologous immunoglobulin light chain sequences. Inone embodiment, the heterologous immunoglobulin light chain sequencesare human immunoglobulin λ and/or κ light chain variable sequences.

In one embodiment, the mouse further comprises a transgene at a locusother than an endogenous immunoglobulin locus, wherein the transgenecomprises a sequence encoding a rearranged or unrearranged heterologousλ or κ light chain sequence (e.g., unrearranged V_(L) and unrearrangedJ_(L), or rearranged VJ) operably linked (for unrearranged) or fused(for rearranged) to an immunoglobulin light chain constant regionsequence. In one embodiment, the heterologous λ or κ light chainsequence is human. In one embodiment, the constant region sequence isselected from rodent, human, and non-human primate. In one embodiment,the constant region sequence is selected from mouse, rat, and hamster.In one embodiment, the transgene comprises a non-immunoglobulin promoterthat drives expression of the light chain sequences. In a specificembodiment, the promoter is a transcriptionally active promoter. In aspecific embodiment, the promoter is a ROSA26 promoter.

In one aspect, a nucleic acid construct is provided, comprising anupstream homology arm and a downstream homology arm, wherein theupstream homology arm comprises a sequence that is identical orsubstantially identical to a human immunoglobulin heavy chain variableregion sequence, the downstream homology arm comprises a sequence thatis identical or substantially identical to a human or mouseimmunoglobulin variable region sequence, and disposed between theupstream and downstream homology arms is a sequence that comprises anucleotide sequence encoding a mouse ADAM6 protein. In a specificembodiment, the sequence encoding the mouse ADAM6 gene is operablylinked with a mouse promoter with which the mouse ADAM6 is linked in awild type mouse.

In one aspect, a targeting vector is provided, comprising (a) anucleotide sequence that is identical or substantially identical to ahuman variable region gene segment nucleotide sequence; and, (b) anucleotide sequence encoding a mouse ADAM6 or ortholog or homolog orfragment thereof that is functional in a mouse.

In one embodiment, the targeting vector further comprises a promoteroperably linked to the sequence encoding the mouse ADAM6. In a specificembodiment, the promoter is a mouse ADAM6 promoter.

In one aspect, a nucleotide construct for modifying a mouseimmunoglobulin heavy chain variable locus is provided, wherein theconstruct comprises at least one site-specific recombinase recognitionsite and a sequence encoding an ADAM6 protein or ortholog or homolog orfragment thereof that is functional in a mouse.

In one aspect, mouse cells and mouse embryos are provided, including butnot limited to ES cells, pluripotent cells, and induced pluripotentcells, that comprise genetic modifications as described herein. Cellsthat are XX and cells that are XY are provided. Cells that comprise anucleus containing a modification as described herein are also provided,e.g., a modification introduced into a cell by pronuclear injection.Cells, embryos, and mice that comprise a virally introduced ADAM6 geneare also provided, e.g., cells, embryos, and mice comprising atransduction construct comprising an ADAM6 gene that is functional inthe mouse.

In one aspect, a genetically modified mouse cell is provided, whereinthe cell lacks a functional endogenous mouse ADAM6 locus, and the cellcomprises an ectopic nucleotide sequence that encodes a mouse ADAM6protein or functional fragment thereof. In one embodiment, the cellfurther comprises a modification of an endogenous immunoglobulin heavychain variable gene sequence. In a specific embodiment, the modificationof the endogenous immunoglobulin heavy chain variable gene sequencecomprises a deletion selected from a deletion of a mouse V_(H) genesegment, a deletion of a mouse D_(H) gene segment, a deletion of a mouseJ_(H) gene segment, and a combination thereof. In a specific embodiment,the mouse comprises a replacement of one or more mouse immunoglobulinV_(H), D_(H), and/or J_(H) sequences with a human immunoglobulinsequence. In a specific embodiment, the human immunoglobulin sequence isselected from a human V_(H), a human V_(L), a human D_(H), a humanJ_(H), a human J_(L), and a combination thereof.

In one embodiment, the cell is a totipotent cell, a pluripotent cell, oran induced pluripotent cell. In a specific embodiment, the cell is amouse ES cell.

In one aspect, a mouse B cell is provided, wherein the mouse B cellcomprises a rearranged immunoglobulin heavy chain gene, wherein the Bcell comprises on a chromosome of the B cell a nucleic acid sequenceencoding an ADAM6 protein or ortholog or homolog or fragment thereofthat is functional in a male mouse. In one embodiment, the mouse B cellcomprises two alleles of the nucleic acid sequence.

In one embodiment, the nucleic acid sequence is on a nucleic acidmolecule (e.g., a B cell chromosome) that is contiguous with therearranged mouse immunoglobulin heavy chain locus.

In one embodiment, the nucleic acid sequence is on a nucleic acidmolecule (e.g., a B cell chromosome) that is distinct from the nucleicacid molecule that comprises the rearranged mouse immunoglobulin heavychain locus.

In one embodiment, the mouse B cell comprises a rearranged non-mouseimmunoglobulin variable gene sequence operably linked to a mouse orhuman immunoglobulin constant region gene, wherein the B cell comprisesa nucleic acid sequence that encodes an ADAM6 protein or ortholog orhomolog or fragment thereof that is functional in a male mouse.

In one aspect, a somatic mouse cell is provided, comprising a chromosomethat comprises a modified immunoglobulin heavy chain locus, and anucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog orfragment thereof that is functional in a male mouse. In one embodiment,the nucleic acid sequence is on the same chromosome as the modifiedimmunoglobulin heavy chain locus. In one embodiment, the nucleic acid ison a different chromosome than the modified immunoglobulin heavy chainlocus. In one embodiment, the somatic cell comprises a single copy ofthe nucleic acid sequence. In one embodiment, the somatic cell comprisesat least two copies of the nucleic acid sequence. In a specificembodiment, the somatic cell is a B cell. In a specific embodiment, thecell is a germ cell. In a specific embodiment, the cell is a stem cell.

In one aspect, a mouse germ cell is provided, comprising a nucleic acidsequence encoding a mouse ADAM6 (or homolog or ortholog or functionalfragment thereof) on a chromosome of the germ cell, wherein the nucleicacid sequence encoding the mouse ADAM6 (or homolog or ortholog orfunctional fragment thereof) is at a position in the chromosome that isdifferent from a position in a chromosome of a wild-type mouse germcell. In one embodiment, the nucleic acid sequence is at a mouseimmunoglobulin locus. In one embodiment, the nucleic acid sequence is onthe same chromosome of the germ cell as a mouse immunoglobulin locus. Inone embodiment, the nucleic acid sequence is on a different chromosomeof the germ cell than the mouse immunoglobulin locus. In one embodiment,the mouse immunoglobulin locus comprises a replacement of at least onemouse immunoglobulin sequence with at least one non-mouse immunoglobulinsequence. In a specific embodiment, the at least one non-mouseimmunoglobulin sequence is a human immunoglobulin sequence.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a mouse as described herein is provided. In a specificembodiment, the cell is a mouse embryonic stem (ES) cell.

In one aspect, a tissue derived from a mouse as described herein isprovided. In one embodiment, the tissue is derived from spleen, lymphnode or bone marrow of a mouse as described herein.

In one aspect, a nucleus derived from a mouse as described herein isprovided. In one embodiment, the nucleus is from a diploid cell that isnot a B cell.

In one aspect, a nucleotide sequence encoding an immunoglobulin variableregion made in a mouse as described herein is provided.

In one aspect, an immunoglobulin heavy chain or immunoglobulin lightchain variable region amino acid sequence of an antibody made in a mouseas described herein is provided.

In one aspect, an immunoglobulin heavy chain or immunoglobulin lightchain variable region nucleotide sequence encoding a variable region ofan antibody made in a mouse as described herein is provided.

In one aspect, an antibody or antigen-binding fragment thereof (e.g.,Fab, F(ab)₂, scFv) made in a mouse as described herein is provided. Inone aspect, a method for making a genetically modified mouse isprovided, comprising replacing one or more immunoglobulin heavy chaingene segments upstream (with respect to transcription of theimmunoglobulin heavy chain gene segments) of an endogenous ADAM6 locusof the mouse with one or more human immunoglobulin heavy chain genesegments, and replacing one or more immunoglobulin gene segmentsdownstream (with respect to transcription of the immunoglobulin heavychain gene segments) of the ADAM6 locus of the mouse with one or morehuman immunoglobulin heavy chain or light chain gene segments. In oneembodiment, the one or more human immunoglobulin gene segments replacingone or more endogenous immunoglobulin gene segments upstream of anendogenous ADAM6 locus of the mouse include V gene segments. In oneembodiment, the human immunoglobulin gene segments replacing one or moreendogenous immunoglobulin gene segments upstream of an endogenous ADAM6locus of the mouse include V and D gene segments. In one embodiment, theone or more human immunoglobulin gene segments replacing one or moreendogenous immunoglobulin gene segments downstream of an endogenousADAM6 locus of the mouse include J gene segments. In one embodiment, theone or more human immunoglobulin gene segments replacing one or moreendogenous immunoglobulin gene segments downstream of an endogenousADAM6 locus of the mouse include D and J gene segments. In oneembodiment, the one or more human immunoglobulin gene segments replacingone or more endogenous immunoglobulin gene segments downstream of anendogenous ADAM6 locus of the mouse include V, D and J gene segments.

In one embodiment, the one or more immunoglobulin heavy chain genesegments upstream and/or downstream of the ADAM6 gene are replaced in apluripotent, induced pluripotent, or totipotent cell to form agenetically modified progenitor cell; the genetically modifiedprogenitor cell is introduced into a host; and, the host comprising thegenetically modified progenitor cell is gestated to form a mousecomprising a genome derived from the genetically modified progenitorcell. In one embodiment, the host is an embryo. In a specificembodiment, the host is selected from a mouse pre-morula (e.g., 8- or4-cell stage), a tetraploid embryo, an aggregate of embryonic cells, ora blastocyst.

In one aspect, a method for making a genetically modified mouse isprovided, comprising replacing a mouse nucleotide sequence thatcomprises a mouse immunoglobulin gene segment and a mouse ADAM6 (orortholog or homolog or fragment thereof functional in a male mouse)nucleotide sequence with a sequence comprising a human immunoglobulingene segment to form a first chimeric locus, then inserting a sequencecomprising a mouse ADAM6-encoding sequence (or a sequence encoding anortholog or homolog or functional fragment thereof) into the sequencecomprising the human immunoglobulin gene segment to form a secondchimeric locus.

In one embodiment, the second chimeric locus comprises a humanimmunoglobulin heavy chain variable (V_(H)) gene segment. In oneembodiment, the second chimeric locus comprises a human immunoglobulinlight chain variable (V_(L)) gene segment. In a specific embodiment, thesecond chimeric locus comprises a human V_(H) gene segment or a humanV_(L) gene segment operably linked to a human D_(H) gene segment and ahuman J_(H) gene segment. In a further specific embodiment, the secondchimeric locus is operably linked to a third chimeric locus thatcomprises a human C_(H)1 sequence, or a human C_(H)1 and human hingesequence, fused with a mouse C_(H)2+C_(H)3 sequence.

In one aspect, use of a mouse that comprises an ectopic nucleotidesequence comprising a mouse ADAM6 locus or sequence to make a fertilemale mouse is provided, wherein the use comprises mating the mousecomprising the ectopic nucleotide sequence that comprises the mouseADAM6 locus or sequence to a mouse that lacks a functional endogenousmouse ADAM6 locus or sequence, and obtaining a progeny that is a femalecapable of producing progeny having the ectopic ADAM6 locus or sequenceor that is a male that comprises the ectopic ADAM6 locus or sequence,and the male exhibits a fertility that is approximately the same as afertility exhibited by a wild-type male mouse.

In one aspect, use of a mouse as described herein to make animmunoglobulin variable region nucleotide sequence is provided.

In one aspect, use of a mouse as described herein to make a fully humanFab or a fully human F(ab)₂ is provided.

In one aspect, use of a mouse as described herein to make animmortalized cell line is provided.

In one aspect, use of a mouse as described herein to make a hybridoma orquadroma is provided.

In one aspect, use of a mouse as described herein to make a phagelibrary containing human heavy chain variable regions and human lightchain variable regions is provided.

In one aspect, use of a mouse as described herein to generate a variableregion sequence for making a human antibody is provided, comprising (a)immunizing a mouse as described herein with an antigen of interest, (b)isolating a lymphocyte from the immunized mouse of (a), (c) exposing thelymphocyte to one or more labeled antibodies, (d) identifying alymphocyte that is capable of binding to the antigen of interest, and(e) amplifying one or more variable region nucleic acid sequence fromthe lymphocyte thereby generating a variable region sequence.

In one embodiment, the lymphocyte is derived from the spleen of themouse. In one embodiment, the lymphocyte is derived from a lymph node ofthe mouse. In one embodiment, the lymphocyte is derived from the bonemarrow of the mouse.

In one embodiment, the labeled antibody is a fluorophore-conjugatedantibody. In one embodiment, the one or more fluorophore-conjugatedantibodies are selected from an IgM, an IgG, and/or a combinationthereof.

In one embodiment, the lymphocyte is a B cell.

In one embodiment, the one or more variable region nucleic acid sequencecomprises a heavy chain variable region sequence. In one embodiment, theone or more variable region nucleic acid sequence comprises a lightchain variable region sequence. In a specific embodiment, the lightchain variable region sequence is an immunoglobulin κ light chainvariable region sequence. In one embodiment, the one or more variableregion nucleic acid sequence comprises a heavy chain and a κ light chainvariable region sequence.

In one embodiment, use of a mouse as described herein to generate aheavy and a κ light chain variable region sequence for making a humanantibody is provided, comprising (a) immunizing a mouse as describedherein with an antigen of interest, (b) isolating the spleen from theimmunized mouse of (a), (c) exposing B lymphocytes from the spleen toone or more labeled antibodies, (d) identifying a B lymphocyte of (c)that is capable of binding to the antigen of interest, and (e)amplifying a heavy chain variable region nucleic acid sequence and a κlight chain variable region nucleic acid sequence from the B lymphocytethereby generating the heavy chain and κ light chain variable regionsequences.

In one embodiment, use of a mouse as described herein to generate aheavy and a κ light chain variable region sequence for making a humanantibody is provided, comprising (a) immunizing a mouse as describedherein with an antigen of interest, (b) isolating one or more lymphnodes from the immunized mouse of (a), (c) exposing B lymphocytes fromthe one or more lymph nodes to one or more labeled antibodies, (d)identifying a B lymphocyte of (c) that is capable of binding to theantigen of interest, and (e) amplifying a heavy chain variable regionnucleic acid sequence and a κ light chain variable region nucleic acidsequence from the B lymphocyte thereby generating the heavy chain and κlight chain variable region sequences.

In one embodiment, use of a mouse as described herein to generate aheavy and a κ light chain variable region sequence for making a humanantibody is provided, comprising (a) immunizing a mouse as describedherein with an antigen of interest, (b) isolating bone marrow from theimmunized mouse of (a), (c) exposing B lymphocytes from the bone marrowto one or more labeled antibodies, (d) identifying a B lymphocyte of (c)that is capable of binding to the antigen of interest, and (e)amplifying a heavy chain variable region nucleic acid sequence and a κlight chain variable region nucleic acid sequence from the B lymphocytethereby generating the heavy chain and κ light chain variable regionsequences. In various embodiments, the one or more labeled antibodiesare selected from an IgM, an IgG, and/or a combination thereof.

In various embodiments, use of a mouse as described herein to generate aheavy and κ light chain variable region sequence for making a humanantibody is provided, further comprising fusing the amplified heavy andlight chain variable region sequences to human heavy and light chainconstant region sequences, expressing the fused heavy and light chainsequences in a cell, and recovering the expressed heavy and light chainsequences thereby generating a human antibody.

In various embodiments, the human heavy chain constant regions areselected from IgM, IgD, IgA, IgE and IgG. In various specificembodiments, the IgG is selected from an IgG1, an IgG2, an IgG3 and anIgG4. In various embodiments, the human heavy chain constant regioncomprises a C_(H)1, a hinge, a C_(H)2, a C_(H)3, a C_(H)4, or acombination thereof. In various embodiments, the light chain constantregion is an immunoglobulin κ constant region. In various embodiments,the cell is selected from a HeLa cell, a DU145 cell, a Lncap cell, aMCF-7 cell, a MDA-MB-438 cell, a PC3 cell, a T47D cell, a THP-1 cell, aU87 cell, a SHSY5Y (human neuroblastoma) cell, a Saos-2 cell, a Verocell, a CHO cell, a GH3 cell, a PC12 cell, a human retinal cell (e.g., aPER.C6™ cell), and a MC3T3 cell. In a specific embodiment, the cell is aCHO cell.

In one aspect, a method for generating a reverse-chimeric rodent-humanantibody specific against an antigen of interest is provided, comprisingthe steps of immunizing a mouse as described herein with the antigen,isolating at least one cell from the mouse producing a reverse-chimericmouse-human antibody specific against the antigen, culturing at leastone cell producing the reverse-chimeric mouse-human antibody specificagainst the antigen, and obtaining said antibody.

In one embodiment, the reverse-chimeric mouse-human antibody comprises ahuman heavy chain variable domain fused with a mouse or rat heavy chainconstant gene, and a human light chain variable domain fused with amouse or rat or human light chain constant gene.

In one embodiment, culturing at least one cell producing thereverse-chimeric rodent-human antibody specific against the antigen isperformed on at least one hybridoma cell generated from the at least onecell isolated from the mouse.

In one aspect, a method for generating a fully human antibody specificagainst an antigen of interest is provided, comprising the steps ofimmunizing a mouse as described herein with the antigen, isolating atleast one cell from the mouse producing a reverse-chimeric rodent-humanantibody specific against the antigen, generating at least one cellproducing a fully human antibody derived from the reverse-chimericrodent-human antibody specific against the antigen, and culturing atleast one cell producing the fully human antibody, and obtaining saidfully human antibody.

In various embodiments, the at least one cell isolated from the mouseproducing a reverse-chimeric rodent-human antibody specific against theantigen is a splenocyte or a B cell.

In various embodiments, the antibody is a monoclonal antibody.

In various embodiments, immunization with the antigen of interest iscarried out with protein, DNA, a combination of DNA and protein, orcells expressing the antigen.

In one aspect, use of a mouse as described herein to make a nucleic acidsequence encoding an immunoglobulin variable region or fragment thereofis provided. In one embodiment, the nucleic acid sequence is used tomake a human antibody or antigen-binding fragment thereof. In oneembodiment, the mouse is used to make an antigen-binding proteinselected from an antibody, a multi-specific antibody (e.g., abi-specific antibody), an scFv, a bi-specific scFv, a diabody, atriabody, a tetrabody, a V-NAR, a V_(HH), a V_(L), a F(ab), a F(ab)₂, aDVD (i.e., dual variable domain antigen-binding protein), a an SVD(i.e., single variable domain antigen-binding protein), or a bispecificT-cell engager (BiTE).

In one aspect, use of a mouse as described herein to introduce anectopic ADAM6 sequence into a mouse that lacks a functional endogenousmouse ADAM6 sequence is provided, wherein the use comprises mating amouse as described herein with the mouse that lacks the functionalendogenous mouse ADAM6 sequence.

In one aspect, use of genetic material from a mouse as described hereinto make a mouse having an ectopic ADAM6 sequence is provided. In oneembodiment, the use comprises nuclear transfer using a nucleus of a cellof a mouse as described herein. In one embodiment, the use comprisescloning a cell of a mouse as described herein to produce an animalderived from the cell. In one embodiment, the use comprises employing asperm or an egg of a mouse as described herein in a process for making amouse comprising the ectopic ADAM6 sequence.

In one aspect, a method for making a fertile male mouse comprising amodified immunoglobulin heavy chain locus is provided, comprisingfertilizing a first mouse germ cell that comprises a modification of anendogenous immunoglobulin heavy chain locus with a second mouse germcell that comprises an ADAM6 gene or ortholog or homolog or fragmentthereof that is functional in a male mouse; forming a fertilized cell;allowing the fertilized cell to develop into an embryo; and, gestatingthe embryo in a surrogate to obtain a mouse.

In one embodiment, the fertilization is achieved by mating a male mouseand a female mouse. In one embodiment, the female mouse comprises theADAM6 gene or ortholog or homolog or fragment thereof. In oneembodiment, the male mouse comprises the ADAM6 gene or ortholog orhomolog or fragment thereof.

In one aspect, use of a nucleic acid sequence encoding a mouse ADAM6protein or an ortholog or homolog thereof or a functional fragment ofthe corresponding ADAM6 protein for restoring or enhancing the fertilityof a mouse having a genome comprising a modification of animmunoglobulin heavy chain locus is provided, wherein the modificationreduces or eliminates endogenous ADAM6 function.

In one embodiment, the nucleic acid sequence is integrated into thegenome of the mouse at an ectopic position. In one embodiment, thenucleic acid sequence is integrated into the genome of the mouse at anendogenous immunoglobulin locus. In a specific embodiment, theendogenous immunoglobulin locus is a heavy chain locus. In oneembodiment, the nucleic acid sequence is integrated into the genome ofthe mouse at a position other than an endogenous immunoglobulin locus.

In one aspect, use of the mouse as described herein for the manufactureof a medicament (e.g., an antigen-binding protein), or for themanufacture of a sequence encoding a variable sequence of a medicament(e.g., an antigen-binding protein), for the treatment of a human diseaseor disorder is provided.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows a general illustration, not to scale, of direct genomicreplacement of about three megabases (Mb) of a mouse immunoglobulinheavy chain variable gene locus (closed symbols) with about one megabase(Mb) of the human immunoglobulin heavy chain variable gene locus (opensymbols).

FIG. 1B shows a general illustration, not to scale, of direct genomicreplacement of about three megabases (Mb) of a mouse immunoglobulin κlight chain variable gene locus (closed symbols) with about 0.5megabases (Mb) of the first, or proximal, of two nearly identicalrepeats of a human immunoglobulin κ light chain variable gene locus(open symbols).

FIG. 2A shows a detailed illustration, not to scale, of three initialsteps (A-C) for direct genomic replacement of a mouse immunoglobulinheavy chain variable gene locus that results in deletion of all mouseV_(H), D_(H) and J_(H) gene segments and replacement with three humanV_(H), all human D_(H) and J_(H) gene segments. A targeting vector for afirst insertion of human immunoglobulin heavy chain gene segments isshown (3hV_(H) BACvec) with a 67 kb 5′ mouse homology arm, a selectioncassette (open rectangle), a site-specific recombination site (opentriangle), a 145 kb human genomic fragment and an 8 kb 3′ mouse homologyarm. Human (open symbols) and mouse (closed symbols) immunoglobulin genesegments, additional selection cassettes (open rectangles) andsite-specific recombination sites (open triangles) inserted fromsubsequent targeting vectors are shown.

FIG. 2B shows a detailed illustration, not to scale, of six additionalsteps (D-I) for direct genomic replacement of a mouse immunoglobulinheavy chain variable gene locus that results in the insertion of 77additional human V_(H) gene segments and removal of a final selectioncassette. A targeting vector for insertion of additional human V_(H)gene segments (18hV_(H) BACvec) to the initial insertion of human heavychain gene segments (3hV_(H)-CRE Hybrid Allele) is shown with a 20 kb 5′mouse homology arm, a selection cassette (open rectangle), a 196 kbhuman genomic fragment and a 62 kb human homology arm that overlaps withthe 5′ end of the initial insertion of human heavy chain gene segmentswhich is shown with a site-specific recombination site (open triangle)located 5′ to the human gene segments. Human (open symbols) and mouse(closed symbols) immunoglobulin gene segments and additional selectioncassettes (open rectangles) inserted by subsequent targeting vectors areshown.

FIG. 2C shows a detailed illustration, not to scale, of three initialsteps (A-C) for direct genomic replacement of a mouse immunoglobulin κlight chain variable gene locus that results in deletion of all mouseVκ, and Jκ gene segments (Igκ-CRE Hybrid Allele). Selection cassettes(open rectangles) and site-specific recombination sites (open triangles)inserted from the targeting vectors are shown.

FIG. 2D shows a detailed illustration, not to scale, of five additionalsteps (D-H) for direct genomic replacement of a mouse immunoglobulin κlight chain variable gene locus that results in the insertion of allhuman Vκ and Jκ gene segments of the proximal repeat and deletion of afinal selection cassette (40hVκdHyg Hybrid Allele). Human (open symbols)and mouse (closed symbols) immunoglobulin gene segments and additionalselection cassettes (open rectangles) inserted by subsequent targetingvectors are shown.

FIG. 3A shows a general illustration, not to scale, of a screeningstrategy including the locations of quantitative PCR (qPCR) primer/probesets to detect insertion of human heavy chain gene sequences and loss ofmouse heavy chain gene sequences in targeted embryonic stem (ES) cells.The screening strategy in ES cells and mice for a first human heavy geneinsertion is shown with qPCR primer/probe sets for the deleted region(“loss” probes C and D), the region inserted (“hIgH” probes G and H) andflanking regions (“retention” probes A, B, E and F) on an unmodifiedmouse chromosome (top) and a correctly targeted chromosome (bottom).

FIG. 3B shows a representative calculation of observed probe copy numberin parental and modified ES cells for a first insertion of humanimmunoglobulin heavy chain gene segments. Observed probe copy number forprobes A through F were calculated as 2/2ΔΔCt. ΔΔCt is calculated asave[ΔCt(sample)−medΔCt(control)] where ΔCt is the difference in Ctbetween test and reference probes (between 4 and 6 reference probesdepending on the assay). The term medΔCt(control) is the median ΔCt ofmultiple (>60) non-targeted DNA samples from parental ES cells. Eachmodified ES cell clone was assayed in sextuplicate. To calculate copynumbers of IgH probes G and H in parental ES cells, these probes wereassumed to have copy number of 1 in modified ES cells and a maximum Ctof 35 was used even though no amplification was observed.

FIG. 3C shows a representative calculation of copy numbers for four miceof each genotype calculated using only probes D and H. Wild-type mice:WT Mice; Mice heterozygous for a first insertion of human immunoglobulingene segments: HET Mice; Mice homozygous for a first insertion of humanimmunoglobulin gene segments: Homo Mice.

FIG. 4A shows a detailed illustration, not to scale, of the three stepsemployed for construction of a 3hV_(H) BACvec by bacterial homologousrecombination (BHR). Human (open symbols) and mouse (closed symbols)immunoglobulin gene segments, selection cassettes (open rectangles) andsite-specific recombination sites (open triangles) inserted fromtargeting vectors are shown.

FIG. 4B shows pulse-field gel electrophoresis (PFGE) of three BAC clones(B1, B2 and B3) after NotI digestion. Markers M1, M2 and M3 are lowrange, mid range and lambda ladder PFG markers, respectively (NewEngland BioLabs, Ipswich, Mass.).

FIG. 5A shows a schematic illustration, not to scale, of sequentialmodifications of a mouse immunoglobulin heavy chain locus withincreasing amounts of human immunoglobulin heavy chain gene segments.Homozygous mice were made from each of the three different stages ofheavy chain humanization. Open symbols indicate human sequence; closedsymbols indicate mouse sequence.

FIG. 5B shows a schematic illustration, not to scale, of sequentialmodifications of a mouse immunoglobulin κ light chain locus withincreasing amounts of human immunoglobulin κ light chain gene segments.Homozygous mice were made from each of the three different stages of κlight chain humanization. Open symbols indicate human sequence; closedsymbols indicate mouse sequence.

FIG. 6 shows FACS dot plots of B cell populations in wild type andVELOCIMMUNE® humanized mice. Cells from spleen (top row, third row fromtop and bottom row) or inguinal lymph node (second row from top) of wildtype (wt), VELOCIMMUNE® 1 (V1), VELOCIMMUNE® 2 (V2) or VELOCIMMUNE® 3(V3) mice were stained for surface IgM expressing B cells (top row, andsecond row from top), surface immunoglobulin containing either κ or λlight chains (third row from top) or surface IgM of specific haplotypes(bottom row), and populations separated by FACS.

FIG. 7A shows representative heavy chain CDR3 sequences of randomlyselected VELOCIMMUNE® antibodies around the V_(H)-D_(H)-J_(H) (CDR3)junction, demonstrating junctional diversity and nucleotide additions.Heavy chain CDR3 sequences are grouped according to D_(H) gene segmentusage, the germline of which is provided above each group in bold. V_(H)gene segments for each heavy chain CDR3 sequence are noted withinparenthesis at the 5′ end of each sequence (e.g., 3-72 is humanV_(H)3-72). J_(H) gene segments for each heavy chain CDR3 are notedwithin parenthesis at the 3′ end of each sequence (e.g., 3 is human 43).SEQ ID NOs for each sequence shown are as follows proceeding from top tobottom: SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ IDNO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ IDNO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ IDNO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39.

FIG. 7B shows representative light chain CDR3 sequences of randomlyselected VELOCIMMUNE® antibodies around the Vκ-Jκ (CDR3) junction,demonstrating junctional diversity and nucleotide additions. Vκ genesegments for each light chain CDR3 sequence are noted within parenthesisat the 5′ end of each sequence (e.g., 1-6 is human Vκ1-6). Jκ genesegments for each light chain CDR3 are noted within parenthesis at the3′ end of each sequence (e.g., 1 is human Jκ1). SEQ ID NOs for eachsequence shown are as follows proceeding from top to bottom: SEQ IDNO:40; SEQ ID NO:41; SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; SEQ IDNO:45; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ IDNO:50; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ IDNO:55; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58.

FIG. 8 shows somatic hypermutation frequencies of heavy and light chainsof VELOCIMMUNE® antibodies scored (after alignment to matching germlinesequences) as percent of sequences changed at each nucleotide (NT; leftcolumn) or amino acid (AA; right column) position among sets of 38(unimmunized IgM), 28 (unimmunized IgG), 32 (unimmunized Igκ from IgG),36 (immunized IgG) or 36 (immunized Igκ from IgG) sequences. Shaded barsindicate the locations of CDRs.

FIG. 9A shows levels of serum immunoglobulin for IgM and IgG isotypes inwild type (open bars) or VELOCIMMUNE® mice (closed bars).

FIG. 9B shows levels of serum immunoglobulin for IgA isotype in wildtype (open bars) or VELOCIMMUNE® mice (closed bars).

FIG. 9C shows levels of serum immunoglobulin for IgE isotype in wildtype (open bars) or VELOCIMMUNE® mice (closed bars).

FIG. 10A shows antigen-specific IgG titers against interleukin-6receptor (IL-6R) of serum from seven VELOCIMMUNE® (VI) and five wildtype (WT) mice after two (bleed 1) or three (bleed 2) rounds ofimmunization with the ectodomain of IL-6R.

FIG. 10B shows anti-IL-6R-specific IgG isotype-specific titers fromseven VELOCIMMUNE® (VI) and five wild type (WT) mice.

FIG. 11A shows the affinity distribution of anti-interleukin-6 receptormonoclonal antibodies generated in VELOCIMMUNE® mice.

FIG. 11B shows the antigen-specific blocking of anti-interleukin-6receptor monoclonal antibodies generated in VELOCIMMUNE® (VI) and wildtype (WT) mice.

FIG. 12 shows a schematic illustration, not to scale, of mouse ADAM6aand ADAM6b genes in a mouse immunoglobulin heavy chain locus. Atargeting vector (mADAM6 Targeting Vector) used for insertion of mouseADAM6a and ADAM6b into a humanized endogenous heavy chain locus is shownwith a selection cassette (HYG: hygromycin) flanked by site-specificrecombination sites (Frt) including engineered restriction sites on the5′ and 3′ ends.

FIG. 13 shows a schematic illustration, not to scale, of a human ADAM6pseudogene (hADAM6Ψ) located between human heavy chain variable genesegments 1-2 (V_(H)1-2) and 6-1 (V_(H)6-1). A targeting vector forbacterial homologous recombination (hADAM6Ψ Targeting Vector) to deletea human ADAM6 pseudogene and insert unique restriction sites into ahuman heavy chain locus is shown with a selection cassette (NEO:neomycin) flanked by site-specific recombination sites (IoxP) includingengineered restriction sites on the 5′ and 3′ ends. An illustration, notto scale, of the resulting targeted humanized heavy chain locuscontaining a genomic fragment that encodes for the mouse ADAM6a andADAM6b genes including a selection cassette flanked by site-specificrecombination sites is shown.

FIG. 14A shows FACS contour plots of lymphocytes gated on singlets forsurface expression of IgM and B220 in the bone marrow for micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κ lightchain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)). Percentage ofimmature (B220^(int) IgM⁺) and mature (B220^(high)IgM⁺) B cells is notedin each contour plot.

FIG. 14B shows the total number of immature (B220^(int)IgM⁺) and mature(B220^(high)IgM⁺) B cells in the bone marrow isolated from femurs ofmice homozygous for human heavy and human κ light chain variable geneloci (H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κlight chain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)).

FIG. 15A shows FACS contour plots of CD19⁺-gated B cells for surfaceexpression of c-kit and CD43 in the bone marrow for mice homozygous forhuman heavy and human κ light chain variable gene loci (H^(+/+)κ^(+/+))and mice homozygous for human heavy and human κ light chain variablegene loci having an ectopic mouse genomic fragment encoding mouse ADAM6genes (H^(+/+)A6^(res)κ^(+/+)). Percentage of pro-B (CD19⁺CD43⁺ckit⁺)and pre-B (CD19⁺CD43⁻ckit⁻) cells is noted in the upper right and lowerleft quadrants, respectively, of each contour plot.

FIG. 15B shows the total number of pro-B cells (CD19⁺CD43⁺ckit⁺) andpre-B cells (CD19⁺CD43⁻ckit⁻) in the bone marrow isolated from femurs ofmice homozygous for human heavy and human κ light chain variable geneloci (H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κlight chain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)).

FIG. 16A shows FACS contour plots of lymphocytes gated on singlets forsurface expression of CD19 and CD43 in the bone marrow for micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κ lightchain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)). Percentage ofimmature B (CD19⁺CD43⁻), pre-B (CD19⁺CD43^(int)) and pro-B (CD19⁺CD43⁺)cells is noted in each contour plot.

FIG. 16B shows histograms of immature B (CD19⁺CD43⁻) and pre-B(CD19⁺CD43^(int)) cells in the bone marrow of mice homozygous for humanheavy and human κ light chain variable gene loci (H^(+/+)κ^(+/+)) andmice homozygous for human heavy and human κ light chain variable geneloci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes(H^(+/+)A6^(res)κ^(+/+)).

FIG. 17A shows FACS contour plots of lymphocytes gated on singlets forsurface expression of CD19 and CD3 in splenocytes for mice homozygousfor human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κ lightchain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)). Percentage of B(CD19⁺CD3⁻) and T (CD19⁻CD3⁺) cells is noted in each contour plot.

FIG. 17B shows FACs contour plots for CD19⁺-gated B cells for surfaceexpression of Igλ and Igκ light chain in the spleen of mice homozygousfor human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κ lightchain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)). Percentage of Igκ⁺(upper left quadrant) and Igκ⁺ (lower right quadrant) B cells is notedin each contour plot.

FIG. 17C shows the total number of CD19⁺ B cells in the spleen of micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κ lightchain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)).

FIG. 18A shows FACs contour plots of CD19⁺-gated B cells for surfaceexpression of IgD and IgM in the spleen of mice homozygous for humanheavy and human κ light chain variable gene loci (H^(+/+)κ^(+/+)) andmice homozygous for human heavy and human κ light chain variable geneloci having an ectopic mouse genomic fragment encoding mouse ADAM6 genes(H^(+/+)A6^(res)κ^(+/+)). Percentage of mature B cells(CD19⁺IgD^(high)IgM^(int)) is noted for each contour plot. The arrow onthe right contour plot illustrates the process of maturation for B cellsin relation to IgM and IgD surface expression.

FIG. 18B shows the total number of B cells in the spleen of micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+)) and mice homozygous for human heavy and human κ lightchain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+)) during maturationfrom CD19⁺IgM^(high)IgD^(int) to CD19⁺IgM^(int)IgD^(high).

FIG. 19 shows the antibody titer for first and second bleeds from micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+); n=5) and mice homozygous for human heavy and human κlight chain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+); n=5) that wereimmunized with a human cell surface receptor (Antigen A).

FIG. 20 shows the antibody titer for first and second bleeds from micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+); n=5) and mice homozygous for human heavy and human κlight chain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+); n=10) that wereimmunized with a human antibody specific for a human receptortyrosine-protein kinase (Antigen B).

FIG. 21 shows the antibody titer for first and second bleeds from micehomozygous for human heavy and human κ light chain variable gene loci(H^(+/+)κ^(+/+); n=12) and mice homozygous for human heavy and human κlight chain variable gene loci having an ectopic mouse genomic fragmentencoding mouse ADAM6 genes (H^(+/+)A6^(res)κ^(+/+); n=12) that wereimmunized with a secreted human protein that functions in regulation ofthe TGF-β signaling pathway (Antigen C).

FIG. 22 shows the antibody titer for first and second bleeds from micehomozygous for human heavy and human κ light chain variable gene locihaving an ectopic mouse genomic fragment encoding mouse ADAM6 genes(H^(+/+)A6^(res)κ^(+/+); n=12) that were immunized with a human receptortyrosine kinase (Antigen D).

DETAILED DESCRIPTION OF INVENTION

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference.

The phrase “substantial” or “substantially” when used to refer to anamount of gene segments (e.g., “substantially all” V gene segments)includes both functional and non functional gene segments and include,in various embodiments, e.g., 80% or more, 85% or more, 90% or more, 95%or more 96% or more, 97% or more, 98% or more, or 99% or more of allgene segments; in various embodiments, “substantially all” gene segmentsincludes, e.g., at least 95%, 96%, 97%, 98%, or 99% of functional (i.e.,non-pseudogene) gene segments.

The term “replacement” includes wherein a DNA sequence is placed into agenome of a cell in such a way as to replace a sequence within thegenome with a heterologous sequence (e.g., a human sequence in a mouse),at the locus of the genomic sequence. The DNA sequence so placed mayinclude one or more regulatory sequences that are part of source DNAused to obtain the sequence so placed (e.g., promoters, enhancers, 5′-or 3′-untranslated regions, appropriate recombination signal sequences,etc.). For example, in various embodiments, the replacement is asubstitution of an endogenous sequence for a heterologous sequence thatresults in the production of a gene product from the DNA sequence soplaced (comprising the heterologous sequence), but not expression of theendogenous sequence; the replacement is of an endogenous genomicsequence with a DNA sequence that encodes a protein that has a similarfunction as a protein encoded by the endogenous genomic sequence (e.g.,the endogenous genomic sequence encodes an immunoglobulin gene ordomain, and the DNA fragment encodes one or more human immunoglobulingenes or domains). In various embodiments, an endogenous gene orfragment thereof is replaced with a corresponding human gene or fragmentthereof. A corresponding human gene or fragment thereof is a human geneor fragment that is an ortholog of, a homolog of, or is substantiallyidentical or the same in structure and/or function, as the endogenousgene or fragment thereof that is replaced.

The mouse as a genetic model has been greatly enhanced by transgenic andknockout technologies, which have allowed for the study of the effectsof the directed over-expression or deletion of specific genes. Despiteall of its advantages, the mouse still presents genetic obstacles thatrender it an imperfect model for human diseases and an imperfectplatform to test human therapeutics or make them. First, although about99% of human genes have a mouse homolog (Waterston et al. 2002, Initialsequencing and comparative analysis of the mouse genome, Nature420:520-562), potential therapeutics often fail to cross-react, orcross-react inadequately, with mouse orthologs of the intended humantargets. To obviate this problem, selected target genes can be“humanized,” that is, the mouse gene can be eliminated and replaced bythe corresponding human orthologous gene sequence (e.g., U.S. Pat. No.6,586,251, U.S. Pat. No. 6,596,541 and U.S. Pat. No. 7,105,348,incorporated herein by reference). Initially, efforts to humanize mousegenes by a “knockout-plus-transgenic humanization” strategy entailedcrossing a mouse carrying a deletion (i.e., knockout) of the endogenousgene with a mouse carrying a randomly integrated human transgene (see,e.g., Bril et al., 2006, Tolerance to factor VIII in a transgenic mouseexpressing human factor VIII cDNA carrying an Arg(593) to Cyssubstitution, Thromb Haemost 95:341-347; Homanics et al., 2006,Production and characterization of murine models of classic andintermediate maple syrup urine disease, BMC Med Genet. 7:33; Jamsai etal., 2006, A humanized BAC transgenic/knockout mouse model forHbE/beta-thalassemia, Genomics 88(3):309-15; Pan et al., 2006, Differentrole for mouse and human CD3delta/epsilon heterodimer in preT cellreceptor (preTCR) function:human CD3delta/epsilon heterodimer restoresthe defective preTCR function in CD3gamma- and CD3gammadelta-deficientmice, Mol Immunol 43:1741-1750). But those efforts were hampered by sizelimitations; conventional knockout technologies were not sufficient todirectly replace large mouse genes with their large human genomiccounterparts. A straightforward approach of direct homologousreplacement, in which an endogenous mouse gene is directly replaced bythe human counterpart gene at the same precise genetic location of themouse gene (i.e., at the endogenous mouse locus), is rarely attemptedbecause of technical difficulties. Until now, efforts at directreplacement involved elaborate and burdensome procedures, thus limitingthe length of genetic material that could be handled and the precisionwith which it could be manipulated.

Exogenously introduced human immunoglobulin transgenes rearrange inprecursor B cells in mice (Alt et al., 1985, Immunoglobulin genes intransgenic mice, Trends Genet. 1:231-236). This finding was exploited byengineering mice using the knockout-plus-transgenic approach to expresshuman antibodies (Green et al., 1994, Antigen-specific human monoclonalantibodies from mice engineered with human Ig heavy and light chainYACs, Nat Genet. 7:13-21; Lonberg et al., 1994, Antigen-specific humanantibodies from mice comprising four distinct genetic modifications,Nature 368:856-859; Jakobovits et al., 2007, From XenoMouse technologyto panitumumab, the first fully human antibody product from transgenicmice, Nat Biotechnol 25:1134-1143). The mouse immunoglobulin heavy chainand κ light chain loci were inactivated in these mice by targeteddeletion of small but critical portions of each endogenous locus,followed by introducing human immunoglobulin gene loci as randomlyintegrated large transgenes, as described above, or minichromosomes(Tomizuka et al., 2000, Double trans-chromosomic mice: maintenance oftwo individual human chromosome fragments containing Ig heavy and kappaloci and expression of fully human antibodies, PNAS USA 97:722-727).Such mice represented an important advance in genetic engineering; fullyhuman monoclonal antibodies isolated from them yielded promisingtherapeutic potential for treating a variety of human diseases (Gibsonet al., 2006, Randomized phase III trial results of panitumumab, a fullyhuman anti-epidermal growth factor receptor monoclonal antibody, inmetastatic colorectal cancer, Clin Colorectal Cancer 6:29-31; Jakobovitset al., 2007; Kim et al., 2007, Clinical efficacy of zanolimumab(HuMax-CD4): two Phase II studies in refractory cutaneous T-celllymphoma, Blood 109(11):4655-62; Lonberg, 2005, Human antibodies fromtransgenic animals, Nat Biotechnol 23:1117-1125; Maker et al., 2005,Tumor regression and autoimmunity in patients treated with cytotoxic Tlymphocyte-associated antigen 4 blockade and interleukin 2: a phasestudy, Ann Surg Oncol 12:1005-1016; McClung et al., 2006, Denosumab inpostmenopausal women with low bone mineral density, New Engl J Med354:821-831). But, as discussed above, these mice exhibit compromised Bcell development and immune deficiencies when compared to wild typemice. Such problems potentially limit the ability of the mice to supporta vigorous humoral response and, consequently, generate fully humanantibodies against some antigens. The deficiencies may be due to: (1)inefficient functionality due to the random introduction of the humanimmunoglobulin transgenes and resulting incorrect expression due to alack of upstream and downstream control elements (Garrett et al., 2005,Chromatin architecture near a potential 3′ end of the IgH locus involvesmodular regulation of histone modifications during B-Cell developmentand in vivo occupancy at CTCF sites, Mol Cell Biol 25:1511-1525; Maniset al., 2003, Elucidation of a downstream boundary of the 3′ IgHregulatory region, Mol Immuno/39:753-760; Pawlitzky et al., 2006,Identification of a candidate regulatory element within the 5′ flankingregion of the mouse IgH locus defined by pro-B cell-specifichypersensitivity associated with binding of PU.1, Pax5, and E2A, JImmunol 176:6839-6851); (2) inefficient interspecies interactionsbetween human constant domains and mouse components of the B-cellreceptor signaling complex on the cell surface, which may impairsignaling processes required for normal maturation, proliferation, andsurvival of B cells (Hombach et al., 1990, Molecular components of theB-cell antigen receptor complex of the IgM class, Nature 343:760-762);and (3) inefficient interspecies interactions between soluble humanimmunoglobulins and mouse Fc receptors that might reduce affinityselection (Rao et al., 2002, Differential expression of the inhibitoryIgG Fc receptor FcgammaRIIB on germinal center cells: implications forselection of high-affinity B cells, J Immunol 169:1859-1868) andimmunoglobulin serum concentrations (Brambell et al., 1964, ATheoretical Model of Gamma-Globulin Catabolism, Nature 203:1352-1354;Junghans and Anderson, 1996, The protection receptor for IgG catabolismis the beta2-microglobulin-containing neonatal intestinal transportreceptor, PNAS USA 93:5512-5516; Rao et al., 2002; Hjelm et al., 2006,Antibody-mediated regulation of the immune response, Scand JImmuno/64:177-184; Nimmerjahn and Ravetch, 2007, Fc-receptors asregulators of immunity, Adv Immuno/96:179-204). These deficiencies canbe corrected by in situ humanization of only the variable regions of themouse immunoglobulin loci within their natural locations at theendogenous heavy and light chain loci. This would effectively result inmice that make “reverse chimeric” (i.e., human V:mouse C) antibodieswhich would be capable of normal interactions and selection with themouse environment based on retaining mouse constant regions. Furthersuch reverse chimeric antibodies may be readily reformatted into fullyhuman antibodies for therapeutic purposes.

Genetically modified animals that comprise a replacement at theendogenous immunoglobulin heavy chain locus with heterologous (e.g.,from another species) immunoglobulin sequences can be made inconjunction with replacements at endogenous immunoglobulin light chainloci or in conjunction with immunoglobulin light chain transgenes (e.g.,chimeric immunoglobulin light chain transgenes or fully human fullymouse, etc.). The species from which the heterologous immunoglobulinheavy chain sequences are derived can vary widely; as withimmunoglobulin light chain sequences employed in immunoglobulin lightchain sequence replacements or immunoglobulin light chain transgenes.

Immunoglobulin variable region nucleic acid sequences, e.g., V, D,and/or J segments, are in various embodiments obtained from a human or anon-human animal. Non-human animals suitable for providing V, D, and/orJ segments include, for example bony fish, cartilaginous fish such assharks and rays, amphibians, reptiles, mammals, birds (e.g., chickens).Non-human animals include, for example, mammals. Mammals include, forexample, non-human primates, goats, sheep, pigs, dogs, bovine (e.g.,cow, bull, buffalo), deer, camels, ferrets and rodents and non-humanprimates (e.g., chimpanzees, orangutans, gorillas, marmosets, rhesusmonkeys baboons). Suitable non-human animals are selected from therodent family including rats, mice, and hamsters. In one embodiment, thenon-human animals are mice. As clear from the context, various non-humananimals can be used as sources of variable domains or variable regiongene segments (e.g., sharks, rays, mammals (e.g., camels, rodents suchas mice and rats).

According to the context, non-human animals are also used as sources ofconstant region sequences to be used in connection with variablesequences or segments, for example, rodent constant sequences can beused in transgenes operably linked to human or non-human variablesequences (e.g., human or non-human primate variable sequences operablylinked to, e.g., rodent, e.g., mouse or rat or hamster, constantsequences). Thus, in various embodiments, human V, D, and/or J segmentsare operably linked to rodent (e.g., mouse or rat or hamster) constantregion gene sequences. In some embodiments, the human V, D, and/or Jsegments (or one or more rearranged VDJ or VJ genes) are operably linkedor fused to a mouse, rat, or hamster constant region gene sequence in,e.g., a transgene integrated at a locus that is not an endogenousimmunoglobulin locus.

In a specific embodiment, a mouse is provided that comprises areplacement of V_(H), D_(H), and J_(H) segments at an endogenousimmunoglobulin heavy chain locus with one or more human V_(H), D_(H),and J_(H) segments, wherein the one or more human V_(H), D_(H), andJ_(H) segments are operably linked to an endogenous immunoglobulin heavychain gene; wherein the mouse comprises a transgene at a locus otherthan an endogenous immunoglobulin locus, wherein the transgene comprisesan unrearranged or rearranged human V_(L) and human J_(L) segmentoperably linked to a mouse or rat or human constant region.

A method for a large in situ genetic replacement of the mouse germlineimmunoglobulin variable gene loci with human germline immunoglobulinvariable gene loci while maintaining the ability of the mice to generateoffspring is described. Specifically, the precise replacement of sixmegabases of both the mouse heavy chain and κ light chain immunoglobulinvariable gene loci with their human counterparts while leaving the mouseconstant regions intact is described. As a result, mice have beencreated that have a precise replacement of their entire germlineimmunoglobulin variable repertoire with equivalent human germlineimmunoglobulin variable sequences, while maintaining mouse constantregions. The human variable regions are linked to mouse constant regionsto form chimeric human-mouse immunoglobulin loci that rearrange andexpress at physiologically appropriate levels. The antibodies expressedare “reverse chimeras,” i.e., they comprise human variable regionsequences and mouse constant region sequences. These mice havinghumanized immunoglobulin variable regions that express antibodies havinghuman variable regions and mouse constant regions are calledVELCOIMMUNE® mice.

VELOCIMMUNE® humanized mice exhibit a fully functional humoral immunesystem that is essentially indistinguishable from that of wild-typemice. They display normal cell populations at all stages of B celldevelopment. They exhibit normal lymphoid organ morphology. Antibodysequences of VELOCIMMUNE® mice exhibit normal V(D)J rearrangement andnormal somatic hypermutation frequencies. Antibody populations in thesemice reflect isotype distributions that result from normal classswitching (e.g., normal isotype cis-switching). Immunizing VELOCIMMUNE®mice results in robust humoral immune responses that generate a large,diverse antibody repertoires having human immunoglobulin variabledomains suitable for use as therapeutic candidates. This platformprovides a plentiful source of naturally affinity-matured humanimmunoglobulin variable region sequences for making pharmaceuticallyacceptable antibodies and other antigen-binding proteins.

It is the precise replacement of mouse immunoglobulin variable sequenceswith human immunoglobulin variable sequences that allows for makingVELOCIMMUNE® mice. Yet even a precise replacement of endogenous mouseimmunoglobulin sequences at heavy and light chain loci with equivalenthuman immunoglobulin sequences, by sequential recombineering of verylarge spans of human immunoglobulin sequences, may present certainchallenges due to divergent evolution of the immunoglobulin loci betweenmouse and man. For example, intergenic sequences interspersed within theimmunoglobulin loci are not identical between mice and humans and, insome circumstances, may not be functionally equivalent. Differencesbetween mice and humans in their immunoglobulin loci can still result inabnormalities in humanized mice, particularly when humanizing ormanipulating certain portions of endogenous mouse immunoglobulin heavychain loci. Some modifications at mouse immunoglobulin heavy chain lociare deleterious. Deleterious modifications can include, for example,loss of the ability of the modified mice to mate and produce offspring.

A precise, large-scale, in situ replacement of six megabases of thevariable regions of the mouse heavy and light chain immunoglobulin loci(V_(H)-D_(H)-J_(H) and Vκ-Jκ) with the corresponding 1.4 megabases humangenomic sequences was performed, while leaving the flanking mousesequences intact and functional within the hybrid loci, including allmouse constant chain genes and locus transcriptional control regions(FIG. 1A and FIG. 1B). Specifically, the human V_(H), D_(H), J_(H), Vκand Jκ gene sequences were introduced through stepwise insertion of 13chimeric BAC targeting vectors bearing overlapping fragments of thehuman germline variable loci into mouse ES cells using VELOCIGENE®genetic engineering technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela et al., 2003, High-throughput engineering of the mouse genomecoupled with high-resolution expression analysis, Nat Biotechnol21:652-659).

Humanization of the mouse immunoglobulin genes represents the largestgenetic modification to the mouse genome to date. While previous effortswith randomly integrated human immunoglobulin transgenes have met withsome success (discussed above), direct replacement of the mouseimmunoglobulin genes with their human counterparts dramaticallyincreases the efficiency with which fully-human antibodies can beefficiently generated in otherwise normal mice. Further, such miceexhibit a dramatically increased diversity of fully human antibodiesthat can be obtained after immunization with virtually any antigen, ascompared with mice bearing disabled endogenous loci and fully humanantibody transgenes. Multiple versions of replaced, humanized lociexhibit completely normal levels of mature and immature B cells, incontrast to mice with randomly integrated human transgenes, whichexhibit significantly reduced B cell populations at various stages ofdifferentiation. While efforts to increase the number of human genesegments in human transgenic mice have reduced such defects, theexpanded immunoglobulin repertoires have not altogether correctedreductions in B cell populations as compared to wild-type mice.

Notwithstanding the near wild-type humoral immune function observed inmice with replaced immunoglobulin loci (i.e., VELOCIMMUNE® mice), thereare other challenges encountered when employing a direct replacement ofthe immunoglobulin that is not encountered in some approaches thatemploy randomly integrated transgenes. Differences in the geneticcomposition of the immunoglobulin loci between mice and humans has leadto the discovery of sequences beneficial for the propagation of micewith replaced immunoglobulin gene segments. Specifically, mouse ADAMgenes located within the endogenous immunoglobulin locus are optimallypresent in mice with replaced immunoglobulin loci, due to their role infertility.

Genomic Location and Function of Mouse ADAM6

Male mice that lack the ability to express any functional ADAM6 proteinsurprisingly exhibit a defect in the ability of the mice to mate and togenerate offspring. The mice lack the ability to express a functionalADAM6 protein by virtue of a replacement of all or substantially allmouse immunoglobulin variable region gene segments with human variableregion gene segments. The loss of ADAM6 function results because theADAM6 locus is located within a region of the endogenous mouseimmunoglobulin heavy chain variable region gene locus, proximal to the3′ end of the V_(H) gene segment locus that is upstream of the D_(H)gene segments. In order to breed mice that are homozygous for areplacement of all or substantially all endogenous mouse heavy chainvariable gene segments with human heavy chain variable gene segments, itis generally a cumbersome approach to set up males and females that areeach homozygous for the replacement and await a productive mating.Successful litters are low in frequency and size. Instead, malesheterozygous for the replacement have been employed to mate with femaleshomozygous for the replacement to generate progeny that are heterozygousfor the replacement, then breed a homozygous mouse therefrom. Theinventors have determined that the likely cause of the loss in fertilityin the male mice is the absence in homozygous male mice of a functionalADAM6 protein.

In various aspects, male mice that comprise a damaged (i.e.,nonfunctional or marginally functional) ADAM6 gene exhibit a reductionor elimination of fertility. Because in mice (and other rodents) theADAM6 gene is located in the immunoglobulin heavy chain locus, theinventors have determined that in order to propagate mice, or create andmaintain a strain of mice, that comprise a replaced immunoglobulin heavychain locus, various modified breeding or propagation schemes areemployed. The low fertility, or infertility, of male mice homozygous fora replacement of the endogenous immunoglobulin heavy chain variable genelocus renders maintaining such a modification in a mouse straindifficult. In various embodiments, maintaining the strain comprisesavoiding infertility problems exhibited by male mice homozygous for thereplacement.

In one aspect, a method for maintaining a strain of mouse as describedherein is provided. The strain of mouse need not comprise an ectopicADAM6 sequence, and in various embodiments the strain of mouse ishomozygous or heterozygous for a knockout (e.g., a functional knockout)of ADAM6.

The mouse strain comprises a modification of an endogenousimmunoglobulin heavy chain locus that results in a reduction or loss infertility in a male mouse. In one embodiment, the modification comprisesa deletion of a regulatory region and/or a coding region of an ADAM6gene. In a specific embodiment, the modification comprises amodification of an endogenous ADAM6 gene (regulatory and/or codingregion) that reduces or eliminates fertility of a male mouse thatcomprises the modification; in a specific embodiment, the modificationreduces or eliminates fertility of a male mouse that is homozygous forthe modification.

In one embodiment, the mouse strain is homozygous or heterozygous for aknockout (e.g., a functional knockout) or a deletion of an ADAM6 gene.

In one embodiment, the mouse strain is maintained by isolating from amouse that is homozygous or heterozygous for the modification a cell,and employing the donor cell in host embryo, and gestating the hostembryo and donor cell in a surrogate mother, and obtaining from thesurrogate mother a progeny that comprises the genetic modification. Inone embodiment, the donor cell is an ES cell. In one embodiment, thedonor cell is a pluripotent cell, e.g., an induced pluripotent cell.

In one embodiment, the mouse strain is maintained by isolating from amouse that is homozygous or heterozygous for the modification a nucleicacid sequence comprising the modification, and introducing the nucleicacid sequence into a host nucleus, and gestating a cell comprising thenucleic acid sequence and the host nucleus in a suitable animal. In oneembodiment, the nucleic acid sequence is introduced into a host oocyteembryo.

In one embodiment, the mouse strain is maintained by isolating from amouse that is homozygous or heterozygous for the modification a nucleus,and introducing the nucleus into a host cell, and gestating the nucleusand host cell in a suitable animal to obtain a progeny that ishomozygous or heterozygous for the modification.

In one embodiment, the mouse strain is maintained by employing in vitrofertilization (IVF) of a female mouse (wild-type, homozygous for themodification, or heterozygous for the modification) employing a spermfrom a male mouse comprising the genetic modification. In oneembodiment, the male mouse is heterozygous for the genetic modification.In one embodiment, the male mouse is homozygous for the geneticmodification.

In one embodiment, the mouse strain is maintained by breeding a malemouse that is heterozygous for the genetic modification with a femalemouse to obtain progeny that comprises the genetic modification,identifying a male and a female progeny comprising the geneticmodification, and employing a male that is heterozygous for the geneticmodification in a breeding with a female that is wild-type, homozygous,or heterozygous for the genetic modification to obtain progenycomprising the genetic modification. In one embodiment, the step ofbreeding a male heterozygous for the genetic modification with awild-type female, a female heterozygous for the genetic modification, ora female homozygous for the genetic modification is repeated in order tomaintain the genetic modification in the mouse strain.

In one aspect, a method is provided for maintaining a mouse strain thatcomprises a replacement of an endogenous immunoglobulin heavy chainvariable gene locus with one or more human immunoglobulin heavy chainsequences, comprising breeding the mouse strain so as to generateheterozygous male mice, wherein the heterozygous male mice are bred tomaintain the genetic modification in the strain. In a specificembodiment, the strain is not maintained by any breeding of a homozygousmale with a wild-type female, or a female homozygous or heterozygous forthe genetic modification.

The ADAM6 protein is a member of the ADAM family of proteins, where ADAMis an acronym for A Disintegrin And Metalloprotease. The ADAM family ofproteins is large and diverse, with diverse functions including celladhesion. Some members of the ADAM family are implicated inspermatogenesis and fertilization. For example, ADAM2 encodes a subunitof the protein fertilin, which is implicated in sperm-egg interactions.ADAM3, or cyritestin, appears necessary for sperm binding to the zonapellucida. The absence of either ADAM2 or ADAM3 results in infertility.It has been postulated that ADAM2, ADAM3, and ADAM6 form a complex onthe surface of mouse sperm cells. The human ADAM6 gene, normally foundbetween human V_(H) gene segments V_(H)1-2 and V_(H)6-1, appears to be apseudogene (FIG. 12). In mice, there are two ADAM6 genes—ADAM6a andADAM6b—that are found in an intergenic region between mouse V_(H) andD_(H) gene segments, and in the mouse the ADAM6a and ADAM6b genes areoriented in opposite transcriptional orientation to that of thesurrounding immunoglobulin gene segments (FIG. 12). In mice, afunctional ADAM6 locus is apparently required for normal fertilization.A functional ADAM6 locus or sequence, then, refers to an ADAM6 locus orsequence that can complement, or rescue, the drastically reducedfertilization exhibited in male mice with missing or nonfunctionalendogenous ADAM6 loci.

The position of the intergenic sequence in mice that encodes ADAM6a andADAM6b renders the intergenic sequence susceptible to modification whenmodifying an endogenous mouse heavy chain. When V_(H) gene segments aredeleted or replaced, or when D_(H) gene segments are deleted orreplaced, there is a high probability that a resulting mouse willexhibit a severe deficit in fertility. In order to compensate for thedeficit, the mouse is modified to include a nucleotide sequence thatencodes a protein that will complement the loss in ADAM6 activity due toa modification of the endogenous mouse ADAM6 locus. In variousembodiments, the complementing nucleotide sequence is one that encodes amouse ADAM6a, a mouse ADAM6b, or a homolog or ortholog or functionalfragment thereof that rescues the fertility deficit.

The nucleotide sequence that rescues fertility can be placed at anysuitable position. It can be placed in the intergenic region, or in anysuitable position in the genome (i.e., ectopically). In one embodiment,the nucleotide sequence can be introduced into a transgene that randomlyintegrates into the mouse genome. In one embodiment, the sequence can bemaintained episomally, that is, on a separate nucleic acid rather thanon a mouse chromosome. Suitable positions include positions that aretranscriptionally permissive or active, e.g., a ROSA26 locus (Zambrowiczet al., 1997, PNAS USA 94:3789-3794), a BT-5 locus (Michael et al.,1999, Mech. Dev. 85:35-47), or an Oct4 locus (Wallace et al., 2000,Nucleic Acids Res. 28:1455-1464). Targeting nucleotide sequences totranscriptionally active loci are described, e.g., in U.S. Pat. No.7,473,557, herein incorporated by reference.

Alternatively, the nucleotide sequence that rescues fertility can becoupled with an inducible promoter so as to facilitate optimalexpression in the appropriate cells and/or tissues, e.g., reproductivetissues. Exemplary inducible promoters include promoters activated byphysical (e.g., heat shock promoter) and/or chemical means (e.g., IPTGor Tetracycline).

Further, expression of the nucleotide sequence can be linked to othergenes so as to achieve expression at specific stages of development orwithin specific tissues. Such expression can be achieved by placing thenucleotide sequence in operable linkage with the promoter of a geneexpressed at a specific stage of development. For example,immunoglobulin sequences from one species engineered into the genome ofa host species are place in operable linkage with a promoter sequence ofa CD19 gene (a B cell specific gene) from the host species. Bcell-specific expression at precise developmental stages whenimmunoglobulins are expressed is achieved.

Yet another method to achieve robust expression of an insertednucleotide sequence is to employ a constitutive promoter. Exemplaryconstitutive promoters include SV40, CMV, UBC, EF1A, PGK and CAGG. In asimilar fashion, the desired nucleotide sequence is placed in operablelinkage with a selected constitutive promoter, which provides high levelof expression of the protein(s) encoded by the nucleotide sequence.

The term “ectopic” is intended to include a displacement, or a placementat a position that is not normally encountered in nature (e.g.,placement of a nucleic acid sequence at a position that is not the sameposition as the nucleic acid sequence is found in a wild-type mouse).The term, in various embodiments, is used in the sense of its objectbeing out of its normal, or proper, position. For example, the phrase“an ectopic nucleotide sequence encoding . . . ” refers to a nucleotidesequence that appears at a position at which it is not normallyencountered in the mouse. For example, in the case of an ectopicnucleotide sequence encoding a mouse ADAM6 protein (or an ortholog orhomolog or fragment thereof that provides the same or similar fertilitybenefit on male mice), the sequence can be placed at a differentposition in the mouse's genome than is normally found in a wild-typemouse. In such cases, novel sequence junctions of mouse sequence will becreated by placing the sequence at a different position in the mouse'sgenome than in a wild-type mouse. A functional homolog or ortholog ofmouse ADAM6 is a sequence that confers a rescue of fertility loss (e.g.,loss of the ability of a male mouse to generate offspring by mating)that is observed in an ADAM6^(−/−) mouse. Functional homologs ororthologs include proteins that have at least about 89% identity ormore, e.g., up to 99% identity, to the amino acid sequence of ADAM6aand/or to the amino acid sequence of ADAM6b, and that can complement, orrescue ability to successfully mate, of a mouse that has a genotype thatincludes a deletion or knockout of ADAM6a and/or ADAM6b.

The ectopic position can be anywhere (e.g., as with random insertion ofa transgene containing a mouse ADAM6 sequence), or can be, e.g., at aposition that approximates (but is not precisely the same as) itslocation in a wild-type mouse (e.g., in a modified endogenous mouseimmunoglobulin locus, but either upstream or downstream of its naturalposition, e.g., within a modified immunoglobulin locus but betweendifferent gene segments, or at a different position in a mouse V-Dintergenic sequence). One example of an ectopic placement is placementwithin a humanized immunoglobulin heavy chain locus. For example, amouse comprising a replacement of one or more endogenous V_(H) genesegments with human V_(H) gene segments, wherein the replacement removesan endogenous ADAM6 sequence, can be engineered to have a mouse ADAM6sequence located within a sequence that contains the human V_(H) genesegments. The resulting modification would generate a (ectopic) mouseADAM6 sequence within a human gene sequence, and the (ectopic) placementof the mouse ADAM6 sequence within the human gene sequence canapproximate the position of the human ADAM6 pseudogene (i.e., betweentwo V segments) or can approximate the position of the mouse ADAMSsequence (i.e., within the V-D intergenic region). The resultingsequence junctions created by the joining of a (ectopic) mouse ADAM6sequence within or adjacent to a human gene sequence (e.g., animmunoglobulin gene sequence) within the germline of the mouse would benovel as compared to the same or similar position in the genome of awild-type mouse.

In various embodiments, non-human animals are provided that lack anADAM6 or ortholog or homolog thereof, wherein the lack renders thenon-human animal infertile, or substantially reduces fertility of thenon-human animal. In various embodiments, the lack of ADAM6 or orthologor homolog thereof is due to a modification of an endogenousimmunoglobulin heavy chain locus. A substantial reduction in fertilityis, e.g., a reduction in fertility (e.g., breeding frequency, pups perlitter, litters per year, etc.) of about 50%, 60%, 70%, 80%, 90%, or 95%or more. In various embodiments, the non-human animals are supplementedwith a mouse ADAM6 gene or ortholog or homolog or functional fragmentthereof that is functional in a male of the non-human animal, whereinthe supplemented ADAMS gene or ortholog or homolog or functionalfragment thereof rescues the reduction in fertility in whole or insubstantial part. A rescue of fertility in substantial part is, e.g., arestoration of fertility such that the non-human animal exhibits afertility that is at least 70%, 80%, or 90% or more as compared with anunmodified (i.e., an animal without a modification to the ADAMS gene orortholog or homolog thereof) heavy chain locus.

The sequence that confers upon the genetically modified animal (i.e.,the animal that lacks a functional ADAM6 or ortholog or homolog thereof,due to, e.g., a modification of a immunoglobulin heavy chain locus) is,in various embodiments, selected from an ADAM6 gene or ortholog orhomolog thereof. For example, in a mouse, the loss of ADAM6 function isrescued by adding, in one embodiment, a mouse ADAM6 gene. In oneembodiment, the loss of ADAM6 function in the mouse is rescued by addingan ortholog or homolog of a closely related specie with respect to themouse, e.g., a rodent, e.g., a mouse of a different strain or species, arat of any species, a rodent; wherein the addition of the ortholog orhomolog to the mouse rescues the loss of fertility due to loss of ADAM6function or loss of an ADAM6 gene. Orthologs and homologs from otherspecies, in various embodiments, are selected from a phylogeneticallyrelated species and, in various embodiments, exhibit a percent identitywith the endogenous ADAM6 (or ortholog) that is about 80% or more, 85%or more, 90% or more, 95% or more, 96% or more, or 97% or more; and thatrescue ADAM6-related or (in a non-mouse) ADAM6 ortholog-related loss offertility. For example, in a genetically modified male rat that lacksADAM6 function (e.g., a rat with an endogenous immunoglobulin heavychain variable region replaced with a human immunoglobulin heavy chainvariable region, or a knockout in the rat immunoglobulin heavy chainregion), loss of fertility in the rat is rescued by addition of a ratADAM6 or, in some embodiments, an ortholog of a rat ADAM6 (e.g., anADAM6 ortholog from another rat strain or species, or, in oneembodiment, from a mouse).

Thus, in various embodiments, genetically modified animals that exhibitno fertility or a reduction in fertility due to modification of anucleic acid sequence encoding an ADAM6 protein (or ortholog or homologthereof) or a regulatory region operably linked with the nucleic acidsequence, comprise a nucleic acid sequence that complements, orrestores, the loss in fertility where the nucleic acid sequence thatcomplements or restores the loss in fertility is from a different strainof the same species or from a phylogenetically related species. Invarious embodiments, the complementing nucleic acid sequence is an ADAM6ortholog or homolog or functional fragment thereof. In variousembodiments, the complementing ADAM6 ortholog or homolog or functionalfragment thereof is from a non-human animal that is closely related tothe genetically modified animal having the fertility defect. Forexample, where the genetically modified animal is a mouse of aparticular strain, an ADAM6 ortholog or homolog or functional fragmentthereof can be obtained from a mouse of another strain, or a mouse of arelated species. In one embodiment, where the genetically modifiedanimal comprising the fertility defect is of the order Rodentia, theADAM6 ortholog or homolog or functional fragment thereof is from anotheranimal of the order Rodentia. In one embodiment, the geneticallymodified animal comprising the fertility defect is of a suborderMyomoropha (e.g., jerboas, jumping mice, mouse-like hamsters, hamsters,New World rats and mice, voles, true mice and rats, gerbils, spiny mice,crested rats, climbing mice, rock mice, white-tailed rats, malagasy ratsand mice, spiny dormice, mole rats, bamboo rats, zokors), and the ADAM6ortholog or homolog or functional fragment thereof is selected from ananimal of order Rodentia, or of the suborder Myomorpha.

In one embodiment, the genetically modified animal is from thesuperfamily Dipodoidea, and the ADAM6 ortholog or homolog or functionalfragment thereof is from the superfamily Muroidea. In one embodiment,the genetically modified animal is from the superfamily Muroidea, andthe ADAM6 ortholog or homolog or functional fragment thereof is from thesuperfamily Dipodoidea.

In one embodiment, the genetically modified animal is a rodent. In oneembodiment, the rodent is selected from the superfamily Muroidea, andthe ADAM6 ortholog or homolog is from a different species within thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors); and the ADAM6 ortholog or homolog isselected from a different species of the same family. In a specificembodiment, the genetically modified rodent is selected from a truemouse or rat (family Muridae), and the ADAM6 ortholog or homolog is froma species selected from a gerbil, spiny mouse, or crested rat. In oneembodiment, the genetically modified mouse is from a member of thefamily Muridae, and the ADAM6 ortholog or homolog is from a differentspecies of the family Muridae. In a specific embodiment, the geneticallymodified rodent is a mouse of the family Muridae, and the ADAM6 orthologor homolog is from a rat, gerbil, spiny mouse, or crested rat of thefamily Muridae.

In various embodiments, one or more rodent ADAM6 orthologs or homologsor functional fragments thereof of a rodent in a family restoresfertility to a genetically modified rodent of the same family that lacksan ADAM6 ortholog or homolog (e.g., Cricetidae (e.g., hamsters, NewWorld rats and mice, voles); Muridae (e.g., true mice and rats, gerbils,spiny mice, crested rats)).

In various embodiments, ADAM6 orthologs, homologs, and fragments thereofare assessed for functionality by ascertaining whether the ortholog,homolog, or fragment restores fertility to a genetically modified malenon-human animal that lacks ADAM6 activity (e.g., a rodent, e.g., amouse or rat, that comprises a knockout of ADAM6 or its ortholog). Invarious embodiments, functionality is defined as the ability of a spermof a genetically modified animal lacking an endogenous ADAM6 or orthologor homolog thereof to migrate an oviduct and fertilize an ovum of thesame specie of genetically modified animal.

In various aspects, mice that comprise deletions or replacements of theendogenous heavy chain variable region locus or portions thereof can bemade that contain an ectopic nucleotide sequence that encodes a proteinthat confers similar fertility benefits to mouse ADAM6 (e.g., anortholog or a homolog or a fragment thereof that is functional in a malemouse). The ectopic nucleotide sequence can include a nucleotidesequence that encodes a protein that is an ADAM6 homolog or ortholog (orfragment thereof) of a different mouse strain or a different species,e.g., a different rodent species, and that confers a benefit infertility, e.g., increased number of litters over a specified timeperiod, and/or increased number of pups per litter, and/or the abilityof a sperm cell of a male mouse to traverse through a mouse oviduct tofertilize a mouse egg.

In one embodiment, the ADAM6 is a homolog or ortholog that is at least89% to 99% identical to a mouse ADAM6 protein (e.g., at least 89% to 99%identical to mouse ADAM6a or mouse ADAM6b). In one embodiment, theectopic nucleotide sequence encodes one or more proteins independentlyselected from a protein at least 89% identical to mouse ADAM6a, aprotein at least 89% identical to mouse ADAM6b, and a combinationthereof. In one embodiment, the homolog or ortholog is a rat, hamster,mouse, or guinea pig protein that is or is modified to be about 89% ormore identical to mouse ADAM6a and/or mouse ADAM6b. In one embodiment,the homolog or ortholog is or is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to a mouse ADAM6a and/or mouse ADAM6b.

Ectopic ADAM6 in Humanized Heavy Chain Mice

Developments in gene targeting, e.g., the development of bacterialartificial chromosomes (BACs), now enable the recombination ofrelatively large genomic fragments. BAC engineering has allowed for theability to make large deletions, and large insertions, into mouse EScells.

Mice that make human antibodies have been available for some time now.Although they represent an important advance in the development of humantherapeutic antibodies, these mice display a number of significantabnormalities that limit their usefulness. For example, they displaycompromised B cell development. The compromised development may be dueto a variety of differences between the transgenic mice and wild-typemice.

Human antibodies might not optimally interact with mouse pre B cell or Bcell receptors on the surface of mouse cells that signal for maturation,proliferation, or survival during clonal selection. Fully humanantibodies might not optimally interact with a mouse Fc receptor system;mice express Fc receptors that do not display a one-to-onecorrespondence with human Fc receptors. Finally, various mice that makefully human antibodies do not include all genuine mouse sequences, e.g.,downstream enhancer elements and other locus control elements, which maybe required for wild-type B cell development.

Mice that make fully human antibodies generally comprise endogenousimmunoglobulin loci that are disabled in some way, and human transgenesthat comprise variable and constant immunoglobulin gene segments areintroduced into a random location in the mouse genome. As long as theendogenous locus is sufficiently disabled so as not to rearrange genesegments to form a functional immunoglobulin gene, the goal of makingfully human antibodies in such a mouse can be achieved—albeit withcompromised B cell development.

Although compelled to make fully human antibodies from the humantransgene locus, generating human antibodies in a mouse is apparently anunfavored process. In some mice, the process is so unfavored as toresult in formation of chimeric human variable/mouse constant heavychains (but not light chains) through the mechanism of trans-switching.By this mechanism, transcripts that encode fully human antibodiesundergo isotype switching in trans from the human isotype to a mouseisotype. The process is in trans, because the fully human transgene islocated apart from the endogenous locus that retains an undamaged copyof a mouse heavy chain constant region gene. Although in such micetrans-switching is readily apparent the phenomenon is still insufficientto rescue B cell development, which remains frankly impaired. In anyevent, trans-switched antibodies made in such mice retain fully humanlight chains, since the phenomenon of trans-switching apparently doesnot occur with respect to light chains; trans-switching presumablyrelies on switch sequences in endogenous loci used (albeit differently)in normal isotype switching in cis. Thus, even when mice engineered tomake fully human antibodies select a trans-switching mechanism to makeantibodies with mouse constant regions, the strategy is stillinsufficient to rescue normal B cell development.

A primary concern in making antibody-based human therapeutics is makinga sufficiently large diversity of human immunoglobulin variable regionsequences to identify useful variable domains that specificallyrecognize particular epitopes and bind them with a desirable affinity,usually—but not always—with high affinity. Prior to the development ofVELOCIMMUNE® mice (described herein), there was no indication that miceexpressing human variable regions with mouse constant regions wouldexhibit any significant differences from mice that made human antibodiesfrom a transgene. That supposition, however, was incorrect.

VELOCIMMUNE® mice, which contain a precise replacement of mouseimmunoglobulin variable regions with human immunoglobulin variableregions at the endogenous mouse loci, display a surprising andremarkable similarity to wild-type mice with respect to B celldevelopment. In a surprising and stunning development, VELOCIMMUNE® micedisplayed an essentially normal, wild-type response to immunization thatdiffered only in one significant respect from wild-type mice—thevariable regions generated in response to immunization are fully human.

VELOCIMMUNE® mice contain a precise, large-scale replacement of germlinevariable regions of mouse immunoglobulin heavy chain (IgH) andimmunoglobulin light chain (e.g., κ light chain, Igκ) with correspondinghuman immunoglobulin variable regions, at the endogenous loci. In total,about six megabases of mouse loci are replaced with about 1.5 megabasesof human genomic sequence. This precise replacement results in a mousewith hybrid immunoglobulin loci that make heavy and light chains thathave a human variable regions and a mouse constant region. The precisereplacement of mouse V_(H)-D_(H)-J_(H) and Vκ-Jκ segments leave flankingmouse sequences intact and functional at the hybrid immunoglobulin loci.The humoral immune system of the mouse functions like that of awild-type mouse. B cell development is unhindered in any significantrespect and a rich diversity of human variable regions is generated inthe mouse upon antigen challenge.

VELOCIMMUNE® mice are possible because immunoglobulin gene segments forheavy and κ light chains rearrange similarly in humans and mice, whichis not to say that their loci are the same or even nearly so—clearlythey are not. However, the loci are similar enough that humanization ofthe heavy chain variable gene locus can be accomplished by replacingabout three million base pairs of contiguous mouse sequence thatcontains all the V_(H), D_(H), and J_(H) gene segments with about onemillion bases of contiguous human genomic sequence covering basicallythe equivalent sequence from a human immunoglobulin locus.

In some embodiments, further replacement of certain mouse constantregion gene sequences with human gene sequences (e.g., replacement ofmouse C_(H)1 sequence with human C_(H)1 sequence, and replacement ofmouse C_(L) sequence with human C_(L) sequence) results in mice withhybrid immunoglobulin loci that make antibodies that have human variableregions and partly human constant regions, suitable for, e.g., makingfully human antibody fragments, e.g., fully human Fab′ s. Mice withhybrid immunoglobulin loci exhibit normal variable gene segmentrearrangement, normal somatic hypermutation frequencies, and normalclass switching. These mice exhibit a humoral immune system that isindistinguishable from wild type mice, and display normal cellpopulations at all stages of B cell development and normal lymphoidorgan structures—even where the mice lack a full repertoire of humanvariable region gene segments. Immunizing these mice results in robusthumoral responses that display a wide diversity of variable gene segmentusage.

The precise replacement of mouse germline variable region gene segmentsallows for making mice that have partly human immunoglobulin loci.Because the partly human immunoglobulin loci rearrange, hypermutate, andclass switch normally, the partly human immunoglobulin loci generateantibodies in a mouse that comprise human variable regions. Nucleotidesequences that encode the variable regions can be identified and cloned,then fused (e.g., in an in vitro system) with any sequences of choice,e.g., any immunoglobulin isotype suitable for a particular use,resulting in an antibody or antigen-binding protein derived wholly fromhuman sequences.

Large-scale humanization by recombineering methods were used to modifymouse embryonic stem (ES) cells to precisely replace up to threemegabases of the mouse heavy chain immunoglobulin locus that includedessentially all of the mouse V_(H), D_(H), and J_(H) gene segments withequivalent human gene segments with up to a one megabase human genomicsequence containing some or essentially all human V_(H), D_(H), andJ_(H) gene segments. Up to a one-half megabase segment of the humangenome comprising one of two repeats encoding essentially all human Vκand Jκ gene segments was used to replace a three megabase segment of themouse immunoglobulin κ light chain locus containing essentially all ofthe mouse Vκ and Jκ gene segments.

Mice with such replaced immunoglobulin loci can comprise a disruption ordeletion of the endogenous mouse ADAM6 locus, which is normally foundbetween the 3′-most V_(H) gene segment and the 5′-most D_(H) genesegment at the mouse immunoglobulin heavy chain locus. Disruption inthis region can lead to reduction or elimination of functionality of theendogenous mouse ADAM6 locus. If the 3′-most V_(H) gene segments of thehuman heavy chain repertoire are used in a replacement, an intergenicregion containing a pseudogene that appears to be a human ADAM6pseudogene is present between these V_(H) gene segments, i.e., betweenhuman V_(H)1-2 and V_(H)1-6. However, male mice that comprise this humanintergenic sequence exhibit a reduction in fertility.

Mice are described that comprise the replaced loci as described above,and that also comprise an ectopic nucleic acid sequence encoding a mouseADAM6, where the mice exhibit essentially normal fertility. In oneembodiment, the ectopic nucleic acid sequence comprises a mouse ADAM6aand/or a mouse ADAM6b sequence or functional fragments thereof placedbetween a human V_(H)1-2 and a human V_(H)6-1 at a modified endogenousheavy chain locus. In one embodiment, the ectopic nucleic acid sequenceis SEQ ID NO:3, placed between a human V_(H)1-2 and a human V_(H)6-1 ata modified endogenous heavy chain locus. The direction of transcriptionof the ADAM6 genes of SEQ ID NO:3 are opposite with respect to thedirection of transcription of the surrounding human V_(H) gene segments.Although examples herein show rescue of fertility by placing the ectopicsequence between the indicated human V_(H) gene segments, skilledpersons will recognize that placement of the ectopic sequence at anysuitable transcriptionally-permissive locus in the mouse genome (or evenextrachromosomally) will be expected to similarly rescue fertility in amale mouse.

The phenomenon of complementing a mouse that lacks a functional ADAM6locus with an ectopic sequence that comprises a mouse ADAM6 gene orortholog or homolog or functional fragment thereof is a general methodthat is applicable to rescuing any mice with nonfunctional or minimallyfunctional endogenous ADAM6 loci. Thus, a great many mice that comprisean ADAM6-disrupting modification of the immunoglobulin heavy chain locuscan be rescued with the compositions and methods of the invention.Accordingly, the invention comprises mice with a wide variety ofmodifications of immunoglobulin heavy chain loci that compromiseendogenous ADAM6 function. Some (non-limiting) examples are provided inthis description. In addition to the VELOCIMMUNE® mice described, thecompositions and methods related to ADAM6 can be used in a great manyapplications, e.g., when modifying a heavy chain locus in a wide varietyof ways.

In one aspect, a mouse is provided that comprises an ectopic ADAM6sequence that encodes a functional ADAM6 protein (or ortholog or homologor functional fragment thereof), a replacement of all or substantiallyall mouse V_(H) gene segments with one or more human V_(H) genesegments, a replacement of all or substantially all mouse D_(H) genesegments and J_(H) gene segments with human D_(H) and human J_(H) genesegments; wherein the mouse lacks a C_(H)1 and/or hinge region. In oneembodiment, the mouse makes a single variable domain binding proteinthat is a dimer of immunoglobulin chains selected from: (a) humanV_(H)-mouse C_(H)1-mouse C_(H)2-mouse C_(H)3; (b) human V_(H)-mousehinge-mouse C_(H)2-mouse C_(H)3; and, (c) human V_(H)-mouse C_(H)2-mouseC_(H)3.

In one aspect, the nucleotide sequence that rescues fertility is placedwithin a human immunoglobulin heavy chain variable region sequence(e.g., between human V_(H)1-2 and V_(H)1-6 gene segments) in a mousethat has a replacement of one or more mouse immunoglobulin heavy chainvariable gene segments (mV_(H)'s, mD_(H)'s, and/or mJ_(H)'s) with one ormore human immunoglobulin heavy chain variable gene segments (hV_(H)'s,hD_(H)'s, and/or hJ_(H)'s), and the mouse further comprises areplacement of one or more mouse immunoglobulin κ light chain variablegene segments (mVκ's and/or mJκ's) with one or more human immunoglobulinκ light chain variable gene segments (hVκ's and/or hJκ's).

In one embodiment, the one or more mouse immunoglobulin heavy chainvariable gene segments comprises about three megabases of the mouseimmunoglobulin heavy chain locus. In one embodiment, the one or moremouse immunoglobulin heavy chain variable gene segments comprises atleast 89 V_(H) gene segments, at least 13 D_(H) gene segments, at leastfour J_(H) gene segments or a combination thereof of the mouseimmunoglobulin heavy chain locus. In one embodiment, the one or morehuman immunoglobulin heavy chain variable gene segments comprises aboutone megabase of a human immunoglobulin heavy chain locus. In oneembodiment, the one or more human immunoglobulin heavy chain variablegene segments comprises at least 80 V_(H) gene segments, at least 27D_(H) gene segments, at least six J_(H) gene segments or a combinationthereof of a human immunoglobulin heavy chain locus.

In one embodiment, the one or more mouse immunoglobulin κ light chainvariable gene segments comprises about three megabases of the mouseimmunoglobulin κ light chain locus. In one embodiment, the one or moremouse immunoglobulin κ light chain variable gene segments comprises atleast 137 Vκ gene segments, at least five Jκ gene segments or acombination thereof of the mouse immunoglobulin κ light chain locus. Inone embodiment, the one or more human immunoglobulin κ light chainvariable gene segments comprises about one-half megabase of a humanimmunoglobulin κ light chain locus. In a specific embodiment, the one ormore human immunoglobulin κ light chain variable gene segments comprisesthe proximal repeat (with respect to the immunoglobulin κ constantregion) of a human immunoglobulin κ light chain locus. In oneembodiment, the one or more human immunoglobulin κ light chain variablegene segments comprises at least 40Vκ gene segments, at least five Jκgene segments or a combination thereof of a human immunoglobulin κ lightchain locus.

In one embodiment, the nucleotide sequence is place between two humanimmunoglobulin gene segments. In a specific embodiment, the two humanimmunoglobulin gene segments are heavy chain gene segments. In oneembodiment, the nucleotide sequence is placed between a human V_(H)1-2gene segment and a human V_(H)1-6 gene segment in a VELOCIMMUNE® mouse(U.S. Pat. No. 6,596,541 and U.S. Pat. No. 7,105,348, incorporatedherein by reference). In one embodiment, the VELOCIMMUNE® mouse somodified comprises a replacement of mouse immunoglobulin heavy chainvariable gene segments with at least 80 human V_(H) gene segments, 27human D_(H) gene segments and six human J_(H) gene segments, and areplacement of mouse immunoglobulin κ light chain variable gene segmentswith at least 40 human Vκ gene segments and five human Jκ gene segments.

In one aspect, a functional mouse ADAM6 locus (or ortholog or homolog orfunctional fragment thereof) is present in the midst of human V_(H) genesegments that replace endogenous mouse V_(H) gene segments. In oneembodiment, at least 89 mouse V_(H) gene segments are removed andreplaced with one or more human V_(H) gene segments, and the mouse ADAM6locus is present immediately adjacent to the 3′ end of the human V_(H)gene segments, or between two human V_(H) gene segments. In a specificembodiment, the mouse ADAM6 locus is present between two V_(H) genesegments within about 20 kilo bases (kb) to about 40 kilo bases (kb) ofthe 3′ terminus of the inserted human V_(H) gene segments. In a specificembodiment, the mouse ADAM6 locus is present between two V_(H) genesegments within about 29 kb to about 31 kb of the 3′ terminus of theinserted human V_(H) gene segments. In a specific embodiment, the mouseADAM6 locus is present within about 30 kb of the 3′ terminus of theinserted human V_(H) gene segments. In a specific embodiment, the mouseADAM6 locus is present within about 30, 184 bp of the 3′ terminus of theinserted human VH gene segments. In a specific embodiment, thereplacement includes human V_(H) gene segments V_(H)1-2 and V_(H)6-1,and the mouse ADAM6 locus is present downstream of the V_(H)1-2 genesegment and upstream of the V_(H)6-1 gene segment. In a specificembodiment, the mouse ADAM6 locus is present between a human V_(H)1-2gene segment and a human V_(H)6-1 gene segment, wherein the 5′ end ofthe mouse ADAM6 locus is about 13,848 bp from the 3′ terminus of thehuman V_(H)1-2 gene segment and the 3′ end of the ADAM6 locus is about29,737 bp 5′ of the human V_(H)6-1 gene segment. In a specificembodiment, the mouse ADAM6 locus comprises SEQ ID NO:3 or a fragmentthereof that confers ADAM6 function within cells of the mouse. In aspecific embodiment, the arrangement of human V_(H) gene segments isthen the following (from upstream to downstream with respect todirection of transcription of the human V_(H) gene segments): humanV_(H)1-2-mouse ADAM6 locus-human V_(H)6-1. In a specific embodiment, theADAM6 pseudogene between human V_(H)1-2 and human V_(H)6-1 is replacedwith the mouse ADAM6 locus. In one embodiment, the orientation of one ormore of mouse ADAM6a and mouse ADAM6b of the mouse ADAM6 locus isopposite with respect to direction of transcription as compared with theorientation of the human V_(H) gene segments. Alternatively, the mouseADAM6 locus is present in the intergenic region between the 3′-mosthuman V_(H) gene segment and the 5′-most D_(H) gene segment. This can bethe case whether the 5′-most D_(H) segment is mouse or human.

Similarly, a mouse modified with one or more human V_(L) gene segments(e.g., Vκ or Vλ segments) replacing all or substantially all endogenousmouse V_(H) gene segments can be modified so as to either maintain theendogenous mouse ADAM6 locus, as described above, e.g., by employing atargeting vector having a downstream homology arm that includes a mouseADAM6 locus or functional fragment thereof, or to replace a damagedmouse ADAM6 locus with an ectopic sequence positioned between two humanV_(L) gene segments or between the human V_(L) gene segments and a D_(H)gene segment (whether human or mouse, e.g., Vλ+m/hD_(H)), or a J genesegment (whether human or mouse, e.g., Vκ+J_(H)). In one embodiment, thereplacement includes two or more human V_(L) gene segments, and themouse ADAM6 locus or functional fragment thereof is present between thetwo 3′-most V_(L) gene segments. In a specific embodiment, thearrangement of human V_(L) gene segments is then the following (fromupstream to downstream with respect to direction of transcription of thehuman gene segments): human V_(L)3′-1-mouse ADAM6 locus-human V_(L)3′.In one embodiment, the orientation of one or more of mouse ADAM6a andmouse ADAM6b of the mouse ADAM6 locus is opposite with respect todirection of transcription as compared with the orientation of the humanV_(L) gene segments. Alternatively, the mouse ADAM6 locus is present inthe intergenic region between the 3′-most human V_(L) gene segment andthe 5′-most D_(H) gene segment. This can be the case whether the 5′-mostD_(H) segment is mouse or human.

In one aspect, a mouse is provided with a replacement of one or moreendogenous mouse V_(H) gene segments, and that comprises at least oneendogenous mouse D_(H) gene segment. In such a mouse, the modificationof the endogenous mouse V_(H) gene segments can comprise a modificationof one or more of the 3′-most V_(H) gene segments, but not the 5′-mostD_(H) gene segment, where care is taken so that the modification of theone or more 3′-most V_(H) gene segments does not disrupt or render theendogenous mouse ADAM6 locus nonfunctional. For example, in oneembodiment the mouse comprises a replacement of all or substantially allendogenous mouse V_(H) gene segments with one or more human V_(H) genesegments, and the mouse comprises one or more endogenous D_(H) genesegments and a functional endogenous mouse ADAM6 locus.

In another embodiment, the mouse comprises the modification ofendogenous mouse 3′-most V_(H) gene segments, and a modification of oneor more endogenous mouse D_(H) gene segments, and the modification iscarried out so as to maintain the integrity of the endogenous mouseADAM6 locus to the extent that the endogenous ADAM6 locus remainsfunctional. In one example, such a modification is done in two steps:(1) replacing the 3′-most endogenous mouse V_(H) gene segments with oneor more human V_(H) gene segments employing a targeting vector with anupstream homology arm and a downstream homology arm wherein thedownstream homology arm includes all or a portion of a functional mouseADAM6 locus; (2) then replacing and endogenous mouse D_(H) gene segmentwith a targeting vector having an upstream homology arm that includes aall or a functional portion of a mouse ADAM6 locus.

In various aspects, employing mice that contain an ectopic sequence thatencodes a mouse ADAM6 protein or an ortholog or homolog or functionalhomolog thereof are useful where modifications disrupt the function ofendogenous mouse ADAM6. The probability of disrupting endogenous mouseADAM6 function is high when making modifications to mouse immunoglobulinloci, in particular when modifying mouse immunoglobulin heavy chainvariable regions and surrounding sequences. Therefore, such mice provideparticular benefit when making mice with immunoglobulin heavy chain locithat are deleted in whole or in part, are humanized in whole or in part,or are replaced (e.g., with Vκ or Vλ sequences) in whole or in part.Methods for making the genetic modifications described for the micedescribed below are known to those skilled in the art.

Mice containing an ectopic sequence encoding a mouse ADAM6 protein, or asubstantially identical or similar protein that confers the fertilitybenefits of a mouse ADAM6 protein, are particularly useful inconjunction with modifications to a mouse immunoglobulin heavy chainvariable gene locus that disrupt or delete the endogenous mouse ADAM6sequence. Although primarily described in connection with mice thatexpress antibodies with human variable regions and mouse constantregions, such mice are useful in connection with any geneticmodifications that disrupt endogenous mouse ADAM6 genes. Persons ofskill will recognize that this encompasses a wide variety of geneticallymodified mice that contain modifications of mouse immunoglobulin heavychain variable gene loci. These include, for example, mice with adeletion or a replacement of all or a portion of mouse immunoglobulinheavy chain gene segments, regardless of other modifications.Non-limiting examples are described below.

In some aspects, genetically modified mice are provided that comprise anectopic mouse, rodent, or other ADAM6 gene (or ortholog or homolog orfragment) functional in a mouse, and one or more human immunoglobulinvariable and/or constant region gene segments. In various embodiments,other ADAM6 gene orthologs or homologs or fragments functional in amouse may include sequences from bovine, canine, primate, rabbit orother non-human sequences.

In one aspect, a mouse is provided that comprises an ectopic ADAM6sequence that encodes a functional ADAM6 protein, a replacement of allor substantially all mouse V_(H) gene segments with one or more humanV_(H) gene segments; a replacement of all or substantially all mouseD_(H) gene segments with one or more human D_(H) gene segments; and areplacement of all or substantially all mouse J_(H) gene segments withone or more human J_(H) gene segments.

In one embodiment, the mouse further comprises a replacement of a mouseC_(H)1 nucleotide sequence with a human C_(H)1 nucleotide sequence. Inone embodiment, the mouse further comprises a replacement of a mousehinge nucleotide sequence with a human hinge nucleotide sequence. In oneembodiment, the mouse further comprises a replacement of animmunoglobulin light chain variable locus (V_(L) and J_(L)) with a humanimmunoglobulin light chain variable locus. In one embodiment, the mousefurther comprises a replacement of a mouse immunoglobulin light chainconstant region nucleotide sequence with a human immunoglobulin lightchain constant region nucleotide sequence. In a specific embodiment, theV_(L), J_(L), and C_(L) are immunoglobulin κ light chain sequences. In aspecific embodiment, the mouse comprises a mouse C_(H)2 and a mouseC_(H)3 immunoglobulin constant region sequence fused with a human hingeand a human C_(H)1 sequence, such that the mouse immunoglobulin locirearrange to form a gene that encodes a binding protein comprising (a) aheavy chain that has a human variable region, a human C_(H)1 region, ahuman hinge region, and a mouse C_(H)2 and a mouse C_(H)3 region; and(b) a gene that encodes an immunoglobulin light chain that comprises ahuman variable domain and a human constant region.

In one aspect, a mouse is provided that comprises an ectopic ADAM6sequence that encodes a functional ADAM6 protein, a replacement of allor substantially all mouse V_(H) gene segments with one or more humanV_(L) gene segments, and optionally a replacement of all orsubstantially all D_(H) gene segments and/or J_(H) gene segments withone or more human D_(H) gene segments and/or human J_(H) gene segments,or optionally a replacement of all or substantially all D_(H) genesegments and J_(H) gene segments with one or more human J_(L) genesegments.

In one embodiment, the mouse comprises a replacement of all orsubstantially all mouse V_(H), D_(H), and J_(H) gene segments with oneor more V_(L), one or more D_(H), and one or more J gene segments (e.g.,Jκ or Jλ), wherein the gene segments are operably linked to anendogenous mouse hinge region, wherein the mouse forms a rearrangedimmunoglobulin chain gene that contains, from 5′ to 3′ in the directionof transcription, human V_(L)-human or mouse D_(H)-human or mouseJ-mouse hinge-mouse C_(H)2-mouse C_(H)3. In one embodiment, the J regionis a human Jκ region. In one embodiment, the J region is a human J_(H)region. In one embodiment, the J region is a human Jλ region. In oneembodiment, the human V_(L) region is selected from a human Vλ regionand a human Vκ region.

In specific embodiments, the mouse expresses a single variable domainantibody having a mouse or human constant region and a variable regionderived from a human Vκ, a human D_(H) and a human Jκ; a human Vκ, ahuman D_(H), and a human J_(H); a human Vλ, a human D_(H), and a humanJλ; a human Vλ, a human D_(H), and a human J_(H); a human Vκ, a humanD_(H), and a human Jλ; a human Vλ, a human D_(H), and a human Jκ. Inspecific embodiment, recombination recognition sequences are modified soas to allow for productive rearrangements to occur between recited V, D,and J gene segments or between recited V and J gene segments.

In one aspect, a mouse is provided that comprises an ectopic ADAM6sequence that encodes a functional ADAM6 protein (or ortholog or homologor functional fragment thereof), a replacement of all or substantiallyall mouse V_(H) gene segments with one or more human V_(L) genesegments, a replacement of all or substantially all mouse D_(H) genesegment and J_(H) gene segments with human J_(L) gene segments; whereinthe mouse lacks a C_(H)1 and/or hinge region.

In one embodiment, the mouse lacks a sequence encoding a C_(H)1 domain.In one embodiment, the mouse lacks a sequence encoding a hinge region.In one embodiment, the mouse lacks a sequence encoding a C_(H)1 domainand a hinge region.

In a specific embodiment, the mouse expresses a binding protein thatcomprises a human immunoglobulin light chain variable domain (λ or κ)fused to a mouse C_(H)2 domain that is attached to a mouse C_(H)3domain.

In one aspect, a mouse is provided that comprises an ectopic ADAM6sequence that encodes a functional ADAM6 protein (or ortholog or homologor functional fragment thereof), a replacement of all or substantiallyall mouse V_(H) gene segments with one or more human V_(L) genesegments, a replacement of all or substantially all mouse D_(H) andJ_(H) gene segments with human J_(L) gene segments.

In one embodiment, the mouse comprises a deletion of an immunoglobulinheavy chain constant region gene sequence encoding a C_(H)1 region, ahinge region, a C_(H)1 and a hinge region, or a C_(H)1 region and ahinge region and a C_(H)2 region.

In one embodiment, the mouse makes a single variable domain bindingprotein comprising a homodimer selected from the following: (a) humanV_(L)-mouse C_(H)1-mouse C_(H)2-mouse C_(H)3; (b) human V_(L)-mousehinge-mouse C_(H)2-mouse C_(H)3; (c) human V_(L)-mouse C_(H)2-mouseC_(H)3.

In one aspect, a mouse is provided with a disabled endogenous heavychain immunoglobulin locus, comprising a disabled or deleted endogenousmouse ADAM6 locus, wherein the mouse comprises a nucleic acid sequencethat expresses a human or mouse or human/mouse or other chimericantibody. In one embodiment, the nucleic acid sequence is present on atransgene integrated that is randomly integrated into the mouse genome.In one embodiment, the nucleic acid sequence is on an episome (e.g., achromosome) not found in a wild-type mouse.

In one embodiment, the mouse further comprises a disabled endogenousimmunoglobulin light chain locus. In a specific embodiment, theendogenous immunoglobulin light chain locus is selected from a kappa (κ)and a lambda (λ) light chain locus. In a specific embodiment, the mousecomprises a disabled endogenous κ light chain locus and a disabled λlight chain locus, wherein the mouse expresses an antibody thatcomprises a human immunoglobulin heavy chain variable domain and a humanimmunoglobulin light chain domain. In one embodiment, the humanimmunoglobulin light chain domain is selected from a human κ light chaindomain and a human λ light chain domain.

In one aspect, a genetically modified animal is provided that expressesa chimeric antibody and expresses an ADAM6 protein or ortholog orhomolog thereof that is functional in the genetically modified animal.

In one embodiment, the genetically modified animal is selected from amouse and a rat. In one embodiment, the genetically modified animal is amouse, and the ADAM6 protein or ortholog or homolog thereof is from amouse strain that is a different strain than the genetically modifiedanimal. In one embodiment, the genetically modified animal is a rodentof family Cricetidae (e.g., a hamster, a New World rat or mouse, avole), and the ADAM6 protein ortholog or homolog is from a rodent offamily Muridae (e.g., true mouse or rat, gerbil, spiny mouse, crestedrat). In one embodiment, the genetically modified animal is a rodent ofthe family Muridae, and the ADAM6 protein ortholog or homolog is from arodent of family Cricetidae.

In one embodiment, the chimeric antibody comprises a human variabledomain and a constant region sequence of a rodent. In one embodiment,the rodent is selected from a rodent of the family Cricetidae and arodent of family Muridae, In a specific embodiment, the rodent of thefamily Cricetidae and of the family Muridae is a mouse. In a specificembodiment, the rodent of the family Cricetidae and of the familyMuridae is a rat. In one embodiment, the chimeric antibody comprises ahuman variable domain and a constant domain from an animal selected froma mouse or rat; in a specific embodiment, the mouse or rat is selectedfrom the family Cricetidae and the family Muridae. In one embodiment,the chimeric antibody comprises a human heavy chain variable domain, ahuman light chain variable domain and a constant region sequence derivedfrom a rodent selected from mouse and rat, wherein the human heavy chainvariable domain and the human light chain are cognate. In a specificembodiment, cognate includes that the human heavy chain and the humanlight chain variable domains are from a single B cell that expresses thehuman light chain variable domain and the human heavy chain variabledomain together and present the variable domains together on the surfaceof an individual B cell.

In one embodiment, the chimeric antibody is expressed from animmunoglobulin locus. In one embodiment, the heavy chain variable domainof the chimeric antibody is expressed from a rearranged endogenousimmunoglobulin heavy chain locus. In one embodiment, the light chainvariable domain of the chimeric antibody is expressed from a rearrangedendogenous immunoglobulin light chain locus. In one embodiment, theheavy chain variable domain of the chimeric antibody and/or the lightchain variable domain of the chimeric antibody is expressed from arearranged transgene (e.g., a rearranged nucleic acid sequence derivedfrom an unrearranged nucleic acid sequence integrated into the animal'sgenome at a locus other than an endogenous immunoglobulin locus). In oneembodiment, the light chain variable domain of the chimeric antibody isexpressed from a rearranged transgene (e.g., a rearranged nucleic acidsequence derived from an unrearranged nucleic acid sequence integratedinto the animal's genome at a locus other than an endogenousimmunoglobulin locus).

In a specific embodiment, the transgene is expressed from atranscriptionally active locus, e.g., a ROSA26 locus, e.g., a murine(e.g., mouse) ROSA26 locus.

In one aspect, a non-human animal is provided, comprising a humanizedimmunoglobulin heavy chain locus, wherein the humanized immunoglobulinheavy chain locus comprises a non-human ADAM6 sequence or ortholog orhomolog thereof.

In one embodiment, the non-human animal is a rodent selected from amouse, a rat, and a hamster.

In one embodiment, the non-human ADAM6 ortholog or homolog is a sequencethat is orthologous and/or homologous to a mouse ADAM6 sequence, whereinthe ortholog or homolog is functional in the non-human animal.

In one embodiment, the non-human animal is selected from a mouse, a rat,and a hamster and the ADAM6 ortholog or homolog is from a non-humananimal selected from a mouse, a rat, and a hamster. In a specificembodiment, the non-human animal is a mouse and the ADAM6 ortholog orhomolog is from an animal that is selected from a different mousespecies, a rat, and a hamster. In specific embodiment, the non-humananimal is a rat, and the ADAM6 ortholog or homolog is from a rodent thatis selected from a different rat species, a mouse, and a hamster. In aspecific embodiment, the non-human animal is a hamster, and the ADAM6ortholog or homolog is form a rodent that is selected from a differenthamster species, a mouse, and a rat.

In a specific embodiment, the non-human animal is from the suborderMyomorpha, and the ADAM6 sequence is from an animal selected from arodent of superfamily Dipodoidea and a rodent of the superfamilyMuroidea. In a specific embodiment, the rodent is a mouse of superfamilyMuroidea, and the ADAM6 ortholog or homolog is from a mouse or a rat ora hamster of superfamily Muroidea.

In one embodiment, the humanized heavy chain locus comprises one or morehuman V_(H) gene segments, one or more human D_(H) gene segments and oneor more human J_(H) gene segments. In a specific embodiment, the one ormore human V_(H) gene segments, one or more human D_(H) gene segmentsand one or more human J_(H) gene segments are operably linked to one ormore human, chimeric and/or rodent (e.g., mouse or rat) constant regiongenes. In one embodiment, the constant region genes are mouse. In oneembodiment, the constant region genes are rat. In one embodiment, theconstant region genes are hamster. In one embodiment, the constantregion genes comprise a sequence selected from a hinge, a C_(H)2, aC_(H)3, and a combination thereof. In specific embodiment, the constantregion genes comprise a hinge, a C_(H)2, and a C_(H)3 sequence.

In one embodiment, the non-human ADAM6 sequence is contiguous with ahuman immunoglobulin heavy chain sequence. In one embodiment, thenon-human ADAM6 sequence is positioned within a human immunoglobulinheavy chain sequence. In a specific embodiment, the human immunoglobulinheavy chain sequence comprises a V, D and/or J gene segment.

In one embodiment, the non-human ADAM6 sequence is positioned betweentwo V gene segments. In one embodiment, the non-human ADAM6 sequence isjuxtaposed between a V and a D gene segment. In one embodiment, themouse ADAM6 sequence is positioned between a V and a J gene segment. Inone embodiment, the mouse ADAM6 sequence is juxtaposed between a D and aJ gene segment.

In one aspect, a genetically modified non-human animal is provided,comprising a B cell that expresses a human V_(H) domain cognate with ahuman V_(L) domain from an immunoglobulin locus, wherein the non-humananimal expresses a non-immunoglobulin non-human protein from theimmunoglobulin locus. In one embodiment, the non-immunoglobulinnon-human protein is an ADAM protein. In a specific embodiment, the ADAMprotein is an ADAM6 protein or homolog or ortholog or functionalfragment thereof.

In one embodiment the non-human animal is a rodent (e.g., mouse or rat).In one embodiment, the rodent is of family Muridae. In one embodiment,the rodent is of subfamily Murinae. In a specific embodiment, the rodentof subfamily Murinae is selected from a mouse and a rat.

In one embodiment, the non-immunoglobulin non-human protein is a rodentprotein. In one embodiment, the rodent is of family Muridae. In oneembodiment, the rodent is of subfamily Murinae. In a specificembodiment, the rodent is selected from a mouse, a rat, and a hamster.

In one embodiment, the human V_(H) and V_(L) domains are attacheddirectly or through a linker to an immunoglobulin constant domainsequence. In a specific embodiment, the constant domain sequencecomprises a sequence selected from a hinge, a C_(H)2 a C_(H)3, and acombination thereof. In a specific embodiment, the human V_(L) domain isselected from a Vκ or a Vλ domain.

In one aspect, a genetically modified non-human animal is provided,comprising in its germline a human immunoglobulin sequence, wherein thesperm of a male non-human animal is characterized by an in vivomigration defect. In one embodiment, the in vivo migration defectcomprises an inability of the sperm of the male non-human animal tomigrate from a uterus through an oviduct of a female non-human animal ofthe same species. In one embodiment, the non-human animal lacks anucleotide sequence that encodes and ADAM6 protein or functionalfragment thereof. In a specific embodiment, the ADAM6 protein orfunctional fragment thereof includes an ADAM6a and/or an ADAM6b proteinor functional fragments thereof. In one embodiment, the non-human animalis a rodent. In a specific embodiment, the rodent is selected from amouse, a rat, and a hamster.

In one aspect, a non-human animal is provided, comprising a humanimmunoglobulin sequence contiguous with a non-human sequence thatencodes an ADAM6 protein or ortholog or homolog or functional fragmentthereof. In one embodiment, the non-human animal is a rodent. In aspecific embodiment, the rodent is selected from a mouse, a rat, and ahamster.

In one embodiment, the human immunoglobulin sequence is animmunoglobulin heavy chain sequence. In one embodiment, theimmunoglobulin sequence comprises one or more V_(H) gene segments. Inone embodiment, the human immunoglobulin sequence comprises one or moreD_(H) gene segments. In one embodiment, the human immunoglobulinsequence comprises one or more J_(H) gene segments. In one embodiment,the human immunoglobulin sequence comprises one or more V_(H) genesegments, one or more D_(H) gene segments and one or more J_(H) genesegments.

In one embodiment, the immunoglobulin sequence comprises one or moreV_(H) gene segments have a high frequency in natural human repertoires.In a specific embodiment, the one or more V_(H) gene segments compriseno more than two V_(H) gene segments, no more than three V_(H) genesegments, no more than four V_(H) gene segments, no more than five V_(H)gene segments, no more than six V_(H) gene segments, no more than sevenVH gene segments, no more than eight V_(H) gene segments, no more thannine V_(H) gene segments, no more than 10 V_(H) gene segments, no morethan 11 V_(H) gene segments, no more than 12 V_(H) gene segments, nomore than 13 V_(H) gene segments, no more than 14 V_(H) gene segments,no more than 15 V_(H) gene segments, no more than 16, V_(H) genesegments, no more than 17 V_(H) gene segments, no more than 18 V_(H)gene segments, no more than 19 V_(H) gene segments, no more than 20V_(H) gene segments, no more than 21 V_(H) gene segments, no more than22 V_(H) gene segments or no more than 23 V_(H) gene segments.

In a specific embodiment, the one or more V_(H) gene segments comprisefive V_(H) gene segments. In a specific embodiment, the one or moreV_(H) gene segments comprise 10 V_(H) gene segments. In a specificembodiment, the one or more V_(H) gene segments comprise 15 V_(H) genesegments. In a specific embodiment, the one or more V_(H) gene segmentscomprise 20 V_(H) gene segments.

In various embodiments, the V_(H) gene segments are selected fromV_(H)6-1, V_(H)1-2, V_(H)1-3, V_(H)2-5, V_(H)3-7, V_(H)1-8, V_(H)3-9,V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)1-18, V_(H)3-20,V_(H)3-21, V_(H)3-23, V_(H)1-24, V_(H)2-26, V_(H)4-28, V_(H)3-30,V_(H)4-31, V_(H)3-33, V_(H)4-34, V_(H)3-35, V_(H)3-38, V_(H)4-39,V_(H)3-43, V_(H)1-45, V_(H)1-46, V_(H)3-48, V_(H)3-49, V_(H)5-51,V_(H)3-53, V_(H)1-58, V_(H)4-59, V_(H)4-61, V_(H)3-64, V_(H)3-66,V_(H)1-69, V_(H)2-70, V_(H)3-72, V_(H)3-73 and V_(H)3-74.

In various embodiments, the V_(H) gene segments are selected fromV_(H)1-2, V_(H)1-8, V_(H)1-18, V_(H)1-46, V_(H)1-69, V_(H)3-7, V_(H)3-9,V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-33, V_(H)3-43, V_(H)3-48, V_(H)4-31, V_(H)4-34, V_(H)4-39,V_(H)4-59, V_(H)5-51 and V_(H)6-1.

In various embodiments, the V_(H) gene segments are selected fromV_(H)1-18, V_(H)1-46, V_(H)1-69, V_(H)3-7, V_(H)3-11, V_(H)3-15,V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-33, V_(H)3-48, V_(H)4-34,V_(H)4-39, V_(H)4-59 and V_(H)5-51.

In various embodiments, the V_(H) gene segments are selected fromV_(H)1-18, V_(H)1-69, V_(H)3-7, V_(H)3-11, V_(H)3-15, V_(H)3-21,V_(H)3-23, V_(H)3-30, V_(H)3-43, V_(H)3-48, V_(H)4-39, V_(H)4-59 andV_(H)5-51.

In various embodiments, the V_(H) gene segments are selected fromV_(H)1-18, V_(H)3-11, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)4-39 andV_(H)4-59.

In various embodiments, the V_(H) gene segments are selected fromV_(H)1-18, V_(H)3-21, V_(H)3-23, V_(H)3-30 and V_(H)4-39.

In various embodiments, the V_(H) gene segments are selected fromV_(H)1-18, V_(H)3-23 and V_(H)4-39.

In various embodiments, the V_(H) gene segments are selected fromV_(H)3-21, V_(H)3-23 and V_(H)3-30.

In various embodiments, the V_(H) gene segments are selected fromV_(H)3-23, V_(H)3-30 and V_(H)4-39.

In a specific embodiment, human immunoglobulin sequence comprises atleast 18 V_(H) gene segments, 27 D_(H) gene segments and six J_(H) genesegments. In a specific embodiment, the human immunoglobulin sequencecomprises at least 39 V_(H) gene segments, 27 D_(H) gene segments andsix J_(H) gene segments. In a specific embodiment, the humanimmunoglobulin sequence comprises at least 80 V_(H) gene segments, 27D_(H) gene segments and six J_(H) gene segments.

In one embodiment, the non-human animal is a mouse, and the mousecomprises a replacement of endogenous mouse V_(H) gene segments with oneor more human V_(H) gene segments, wherein the human V_(H) gene segmentsare operably linked to a mouse C_(H) region gene, such that the mouserearranges the human V_(H) gene segments and expresses a reversechimeric immunoglobulin heavy chain that comprises a human V_(H) domainand a mouse C_(H). In one embodiment, 90-100% of unrearranged mouseV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially allof the endogenous mouse V_(H) gene segments are replaced with at leastone unrearranged human V_(H) gene segment. In one embodiment, thereplacement is with at least 19, at least 39, or at least 80 or 81unrearranged human V_(H) gene segments. In one embodiment, thereplacement is with at least 12 functional unrearranged human V_(H) genesegments, at least 25 functional unrearranged human V_(H) gene segments,or at least 43 functional unrearranged human V_(H) gene segments. In oneembodiment, the mouse comprises a replacement of all mouse D_(H) andJ_(H) segments with at least one unrearranged human D_(H) segment and atleast one unrearranged human J_(H) segment. In one embodiment, the atleast one unrearranged human D_(H) segment is selected from 1-1, 1-7,1-26, 2-8, 2-15, 3-3, 3-10, 3-16, 3-22, 5-5, 5-12, 6-6, 6-13, 7-27, anda combination thereof. In one embodiment, the at least one unrearrangedhuman J_(H) segment is selected from 1, 2, 3, 4, 5, 6, and a combinationthereof. In a specific embodiment, the one or more human V_(H) genesegment is selected from a 1-2, 1-8, 1-24, 1-69, 2-5, 3-7, 3-9, 3-11,3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 3-53, 4-31, 4-39, 4-59, 5-51,a 6-1 human V_(H) gene segment, and a combination thereof.

In various embodiments, the human immunoglobulin sequence is in operablelinkage with a constant region in the germline of the non-human animal(e.g., the rodent, e.g., the mouse, rat, or hamster). In one embodiment,the constant region is a human, chimeric human/mouse or chimerichuman/rat or chimeric human/hamster, a mouse, a rat, or a hamsterconstant region. In one embodiment, the constant region is a rodent(e.g., mouse or rat or hamster) constant region. In a specificembodiment, the rodent is a mouse or rat. In various embodiments, theconstant region comprises at least a C_(H)2 domain and a C_(H)3 domain.

In one embodiment, the human immunoglobulin heavy chain sequence islocated at an immunoglobulin heavy chain locus in the germline of thenon-human animal (e.g., the rodent, e.g., the mouse or rat or hamster).In one embodiment, the human immunoglobulin heavy chain sequence islocated at a non-immunoglobulin heavy chain locus in the germline of thenon-human animal, wherein the non-heavy chain locus is atranscriptionally active locus. In a specific embodiment, the non-heavychain locus is a ROSA26 locus.

In various aspects, the non-human animal further comprises a humanimmunoglobulin light chain sequence (e.g., one or more unrearrangedlight chain V and J sequences, or one or more rearranged VJ sequences)in the germline of the non-human animal. In a specific embodiment, theimmunoglobulin light chain sequence is an immunoglobulin κ light chainsequence. In one embodiment, the human immunoglobulin light chainsequence comprises one or more V_(L) gene segments. In one embodiment,the human immunoglobulin light chain sequence comprises one or moreJ_(L) gene segments. In one embodiment, the human immunoglobulin lightchain sequence comprises one or more V_(L) gene segments and one or moreJ_(L) gene segments. In a specific embodiment, the human immunoglobulinlight chain sequence comprises at least 16 Vκ gene segments and five Jκgene segments. In a specific embodiment, the human immunoglobulin lightchain sequence comprises at least 30 Vκ gene segments and five Jκ genesegments. In a specific embodiment, the human immunoglobulin light chainsequence comprises at least 40 Vκ gene segments and five Jκ genesegments. In various embodiments, the human immunoglobulin light chainsequence is in operable linkage with a constant region in the germlineof the non-human animal (e.g., rodent, e.g., mouse or rat or hamster).In one embodiment, the constant region is a human, chimerichuman/rodent, mouse, rat, or hamster constant region. In a specificembodiment, the constant region is a mouse or rat constant region. In aspecific embodiment, the constant region is a mouse κ constant (mCκ)region or a rat κ constant (rCκ) region.

In one embodiment, the non-human animal is a mouse and the mousecomprises a replacement of all or substantially all Vκ and Jκ genesegments with at least six human Vκ gene segments and at least one Jκgene segment. In one embodiment, all or substantially all Vκ and Jκ genesegments are replaced with at least 16 human Vκ gene segments (human Vκ)and at least one Jκ gene segment. In one embodiment, all orsubstantially all Vκ and Jκ gene segments are replaced with at least 30human Vκ gene segments and at least one Jκ gene segment. In oneembodiment, all or substantially all Vκ and Jκ gene segments arereplaced with at least 40 human Vκ gene segments and at least one Jκgene segment. In one embodiment, the at least one Jκ gene segmentcomprises two, three, four, or five human Jκ gene segments.

In one embodiment, the human Vκ gene segments comprise Vκ4-1, Vκ5-2,Vκ7-3, Vκ2-4, Vκ1-5, and Vκ1-6. In one embodiment, the Vκ gene segmentscomprise Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14,Vκ3-15 and Vκ1-16. In one embodiment, the human Vκ gene segmentscomprise Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24,Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, and Vκ2-30. In one embodiment,the human Vκ gene segments comprise Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34,Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, and Vκ2-40.

In a specific embodiment, the Vκ gene segments comprise contiguous humanimmunoglobulin κ gene segments spanning the human immunoglobulin κ lightchain locus from Vκ4-1 through Vκ2-40, and the Jκ gene segments comprisecontiguous gene segments spanning the human immunoglobulin κ light chainlocus from Jκ1 through Jκ5.

In one embodiment, the human immunoglobulin light chain sequence islocated at an immunoglobulin light chain locus in the germline of thenon-human animal. In a specific embodiment, the immunoglobulin lightchain locus in the germline of the non-human animal is an immunoglobulinκ light chain locus. In one embodiment, the human immunoglobulin lightchain sequence is located at a non-immunoglobulin light chain locus inthe germline of the non-human animal that is transcriptionally active.In a specific embodiment, the non-immunoglobulin locus is a ROSA26locus.

In one aspect, a method of making a human antibody is provided, whereinthe human antibody comprises variable domains derived from one or morevariable region nucleic acid sequences encoded in a cell of a non-humananimal as described herein.

In one aspect, a pharmaceutical composition is provided, comprising apolypeptide that comprises antibody or antibody fragment that is derivedfrom one or more variable region nucleic acid sequences isolated from anon-human animal as described herein. In one embodiment, the polypeptideis an antibody. In one embodiment, the polypeptide is a heavy chain onlyantibody. In one embodiment, the polypeptide is a single chain variablefragment (e.g., an scFv).

In one aspect, use of a non-human animal as described herein to make anantibody is provided. In various embodiments, the antibody comprises oneor more variable domains that are derived from one or more variableregion nucleic acid sequences isolated from the non-human animal. In aspecific embodiment, the variable region nucleic acid sequences compriseimmunoglobulin heavy chain gene segments. In a specific embodiment, thevariable region nucleic acid sequences comprise immunoglobulin lightchain gene segments.

EXAMPLES

The following examples are provided so as to describe how to make anduse methods and compositions of the invention, and are not intended tolimit the scope of what the inventors regard as their invention. Unlessindicated otherwise, temperature is indicated in Celsius, and pressureis at or near atmospheric.

Example 1

Humanization of Mouse Immunoglobulin Genes

Human and mouse bacterial artificial chromosomes (BACs) were used toengineer 13 different BAC targeting vectors (BACvecs) for humanizationof the mouse immunoglobulin heavy chain and κ light chain loci. Tables 1and 2 set forth descriptions of the steps performed for construction ofall BACvecs employed for the humanization of mouse immunoglobulin heavychain and κ light chain loci, respectively.

Identification of Human and Mouse BACs.

Mouse BACs that span the 5′ and 3′ ends of the immunoglobulin heavychain and κ light chain loci were identified by hybridization of filtersspotted with BAC library or by PCR screening mouse BAC library DNApools. Filters were hybridized under standard conditions using probesthat corresponded to the regions of interest. Library pools werescreened by PCR using unique primer pairs that flank the targeted regionof interest. Additional PCR using the same primers was performed todeconvolute a given well and isolate the corresponding BAC of interest.Both BAC filters and library pools were generated from 129 SvJ mouse EScells (Incyte Genomics/Invitrogen). Human BACs that cover the entireimmunoglobulin heavy chain and κ light chain loci were identified eitherby hybridization of filters spotted with BAC library (Caltech B, C, or Dlibraries & RPCI-11 library, Research Genetics/Invitrogen) throughscreening human BAC library pools (Caltech library, Invitrogen) by aPCR-based method or by using a BAC end sequence database (Caltech Dlibrary, TIGR).

Construction of BACvecs by Bacterial Homologous Recombination andLigation.

Bacterial homologous recombination (BHR) was performed as described(Valenzuela et al., 2003; Zhang et al., 1998, A new logic for DNAengineering using recombination in Escherichia coli, Nat Genet.20:123-128). In most cases, linear fragments were generated by ligatingPCR-generated homology boxes to cloned cassettes followed by gelisolation of ligation products and electroporation into BHR-competentbacteria harboring the target BAC. After selection on appropriateantibiotic petri dishes, correctly recombined BACs were identified byPCR across both novel junctions followed by restriction analysis onpulsed-field gels (Schwartz and Cantor, 1984, Separation of yeastchromosome-sized DNAs by pulsed field gradient gel electrophoresis, Cell37:67-75) and spot-checking by PCR using primers distributed across thehuman sequences.

A 3hV_(H) BACvec was constructed using three sequential BHR steps forthe initial step of humanization of the immunoglobulin heavy chain locus(FIG. 4A and Table 1). In the first step (Step 1), a cassette wasintroduced into a human parental BAC upstream from the human V_(H)1-3gene segment that contains a region of homology to the mouseimmunoglobulin heavy chain locus (HB1), a gene that confers kanamycinresistance in bacteria and G418 resistance in animals cells (kanR) and asite-specific recombination site (e.g., IoxP). In the second step (Step2), a second cassette was introduced just downstream from the last J_(H)segment that contains a second region of homology to the mouseimmunoglobulin heavy chain locus (HB2) and a gene that confersresistance in bacteria to spectinomycin (specR). This second stepincluded deleting human immunoglobulin heavy chain locus sequencesdownstream from J_(H)6 and the BAC vector chloramphenicol resistancegene (cmR). In the third step (Step 3), the doubly modified human BAC(B1) was then linearized using I-Ceul sites that had been added duringthe first two steps and integrated into a mouse BAC (B2) by BHR throughthe two regions of homology (HB1 and HB2). The drug selections for first(cm/kan), second (spec/kan) and third (cm/kan) steps were designed to bespecific for the desired products. Modified BAC clones were analyzed bypulse-filed gel electrophoresis (PFGE) after digestion with restrictionenzymes to determine appropriate construction (FIG. 4B).

In a similar fashion, 12 additional BACvecs were engineered forhumanization of the heavy chain and κ light chain loci. In someinstances, BAC ligation was performed in lieu of BHR to conjoin twolarge BACs through introduction of rare restriction sites into bothparental BACvecs by BHR along with careful placement of selectablemarkers. This allowed for the survival of the desired ligation productupon selection with specific drug marker combinations. Recombinant BACsobtained by ligation after digestion with rare restriction enzymes wereidentified and screened in a similar fashion to those obtained by BHR(as described above).

TABLE 1 BACvec Step Description Process 3hV_(H) 1 Insert upstream mousehomology box into human BHR proximal BAC CTD-2572o2 2 Insert downstreammouse homology box into human BHR proximal BAC CTD-2572o2 3 Insert3hV_(H)/27hD_(H)/9hJ_(H) into mouse proximal BAC CT7- BHR 302a07 tocreate 3hV_(H) BACvec DC 1 Insert cassette at distal end of mouse IgHlocus using BHR mouse BAC CT7-253i20 18hV_(H) 1 Insert specR marker atdownstream end of 3hV_(H) BHR insertion using human BAC CTD-2572o2 2Insert I-CeuI and Not sites flanking puroR at upstream BHR end of3hV_(H) insertion 3 Insert Not site at downstream end of Rel2-408p02 BACBHR (≈10 kb downstream of V_(H)2-5) 4 Insert I-CeuI site at upstream endof Rel2-408p02 BAC BHR (≈23 kb upstream of V_(H)1-18) 5 Ligate 184 kbfragment from step 4 into 153 kb vector Ligation from step 2 6 Trimhuman homology from CTD-2572o2 BAC BHR deleting ≈85 kb and leaving 65 kbhomology to 3hV_(H) 7 Insert cassette and Not site at distal end ofmouse IgH BHR locus in CT7-253i20 BAC 8 Subclone mouse distal homologyarm for insertion Ligation upstream from human BACs 9 Insert 20 kb mousearm upstream of Rel2-408p02 BHR 10 Swap selection cassette from hygR toneoR to create BHR 18hV_(H) BACvec 39hV_(H) 1 Insert I-CeuI and PI-SceIsites flanking hygR into distal BHR end of human BAC CTD-2534n10 2Insert CmR at proximal end of CTD-2534n10 BAC to BHR allow for selectionfor ligation to RP11-72n10 BAC 3 Insert PI-SceI site into RP11-72n10 BACfor ligation to BHR CTD-2534n10 BAC 4 Insert I-CeuI and AscI sitesflanking puroR at distal end BHR of RP11-72n10 BAC 5 Ligate 161 kbfragment from construct of step 4 into Ligation construct of step 2replacing hygR 6 Insert neoR and AscI site at proximal end of mouse BHRdistal homology arm using CT7-253i20 BAC 7 Insert specR and I-CeuI siteat distal end of mouse BHR distal homology arm 8 Ligate mouse distalhomology arm onto human insert Ligation from step 5 9 Swap selectioncassette from neo to hyg using UbCp BHR and pA as homology boxes tocreate 39hV_(H) BACvec 53hV_(H) 1 Insert specR at proximal end of humanCTD-3074b5 BHR BAC 2 Insert AscI site at distal end of human CTD-3074b5BHR BAC 3 Insert hygR and AscI site at proximal end of mouse BHR distalhomology arm using CT7-253i20 BAC 4 Ligate mouse distal homology armonto construct from Ligation step 2 5 Swap selection cassette from hygto neo using UbCp BHR and pA as homology boxes to create 53hV_(H) BACvec70hV_(H) 1 Insert PI-SceI and I-CeuI sites flanking spec at distal BHRend of human CTD-2195p5 BAC 2 Insert I-CeuI site at proximal end ofRP11-926p12 BAC BHR for ligation to CTD-2195p5 BAC 3 Insert PI-SceI andAscI sites at distal end of RP11- BHR 926p12 BAC for ligation of mousearm 4 Ligate mouse distal homology arm onto construct from Ligation step3 5 Ligate mouse distal homology arm and hIgH fragment Ligation fromRP11-926p12 BAC onto CTD-2195p5 BAC to create 70 hV_(H) BACvec 80hV_(H)1 Insert I-CeuI and AscI sites flanking hygR at distal end BHR ofCTD-2313e3 BAC 2 Ligate mouse distal homology arm onto human CTD-Ligation 2313e3 BAC from step 1 to create 80hV_(H) BACvec

TABLE 2 BACvec Step Description Process Igκ-PC 1 Insert loxP site withinmouse J-C intron using CT7- BHR 254m04 BAC Igκ-DC 1 Insert loxP site atdistal end of mouse Igκ locus using BHR CT7-302g12 BAC 6hVκ 1 InsertPI-SceI site ≈400 bp downstream from hJκ5 in BHR CTD-2366j12 BAC 2Insert I-CeuI and AscI sites flanking hygR at distal BHR end ofCTD-2366j12 BAC 3 Insert I-CeuI and PI-SceI sites flanking puroR BHRdownstream from mJκ using CT7-254m04 BAC 4 Insert hIgVκ/Jκ upstream frommouse Enhκ/Cκ using Ligation construct from step 3 5 Replace cmR inconstruct of step 4 with specR BHR 6 Insert Neo selection cassette atdistal end of mouse BHR Igκ locus using CT7-302g12 BAC 7 Ligate mousedistal homology arm upstream of Ligation human insert in construct ofstep 6 to create 6hVκ BACvec 16hVκ 1 Insert NeoR at distal end ofRP11-1061b13 BAC BHR 2 Replace cmR in construct of step 1 with specR BHR3 Insert Hyg selection cassette at distal end of mouse BHR Igκ locususing CT7-302g12 BAC 4 Ligate mouse distal homology arm upstream ofLigation human insert from construct of step 2 to create 16hVκ BACvec30hVκ 1 Insert HygR at distal end of RP11-99g6 BAC BHR 2 Replace cmR inconstruct of step 1 with specR BHR 3 Insert Neo selection cassette atdistal end of mouse BHR Igκ locus using CT7-302g12 BAC 4 Ligate mousedistal homology arm upstream of Ligation human insert from construct ofstep 2 to create 30hVκ BACvec 40hVκ 1 Insert NeoR at distal end of hIgHlocus in CTD- BHR 2559d6 BAC 2 Replace cmR in construct of step 1 withspecR BHR 3 Ligate mouse distal homology arm upstream of Ligation humaninsert in construct of step 2 to create 40hVκ BACvec

Modification of Embryonic Stem (ES) Cells and Generation of Mice.

ES cell (F1H4) targeting was performed using the VELOCIGENE® geneticengineering method as described (Valenzuela et al., 2003). Derivation ofmice from modified ES cells by either blastocyst (Valenzuela et al.,2003) or 8-cell injection (Poueymirou at al., 2007, F0 generation micefully derived from gene-targeted embryonic stem cells allowing immediatephenotypic analyses, Nat Biotechnol 25:91-99) was as described. TargetedES cells and mice were confirmed by screening DNA from ES cells or micewith unique sets of probes and primers in a PCR based assay (e.g., FIGS.3A, 3B and 3C). All mouse studies were overseen and approved byRegeneron's Institutional Animal Care and Use Committee (IACUC).

Karyotype Analysis and Fluorescent In Situ Hybridization (FISH).

Karyotype Analysis was performed by Coriell Cell Repositories (CoriellInstitute for Medical Research, Camden, N.J.). FISH was performed ontargeted ES cells as described (Valenzuela et al., 2003). Probescorresponding to either mouse BAC DNA or human BAC DNA were labeled bynick translation (Invitrogen) with the fluorescently labeled dUTPnucleotides spectrum orange or spectrum green (Vysis).

Immunoglobulin Heavy Chain Variable Gene Locus.

Humanization of the variable region of the heavy chain locus wasachieved in nine sequential steps by the direct replacement of aboutthree million base pairs (Mb) of contiguous mouse genomic sequencecontaining all V_(H), D_(H) and J_(H) gene segments with about one Mb ofcontiguous human genomic sequence containing the equivalent human genesegments (FIG. 1A and Table 1) using VELOCIGENE® genetic engineeringtechnology (see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al.,2003).

The intron between J_(H) gene segments and constant region genes (theJ-C intron) contains a transcriptional enhancer (Neuberger, 1983,Expression and regulation of immunoglobulin heavy chain gene transfectedinto lymphoid cells, EMBO J. 2:1373-1378) followed by a region of simplerepeats required for recombination during isotype switching (Kataoka etal., 1980, Rearrangement of immunoglobulin gamma 1-chain gene andmechanism for heavy-chain class switch, PNAS USA 77:919-923). Thejunction between human V_(H)-D_(H)-J_(H) region and the mouse C_(H)region (the proximal junction) was chosen to maintain the mouse heavychain intronic enhancer and switch domain in order preserve bothefficient expression and class switching of the humanized heavy chainlocus within the mouse. The exact nucleotide position of this andsubsequent junctions in all the replacements was possible by use of theVELOCIGENE® genetic engineering method (supra), which employed bacterialhomologous recombination driven by synthesized oligonucleotides. Thus,the proximal junction was placed about 200 bp downstream from the lastJ_(H) gene segment and the distal junction was placed several hundredupstream of the most 5′ V_(H) gene segment of the human locus and about9 kb downstream from the mouse V_(H)1-86 gene segment, also known asJ558.55. The mouse V_(H)1-86 (J558.55) gene segment is the most distalheavy chain variable gene segment, reported to be a pseudogene inC57BL/6 mice, but potentially active, albeit with a poor RSS sequence,in the targeted 129 allele. The distal end of the mouse heavy chainlocus reportedly may contain control elements that regulate locusexpression and/or rearrangement (Pawlitzky et al., 2006).

A first insertion of human immunoglobulin DNA sequence into the mousewas achieved using 144 kb of the proximal end of the human heavy chainlocus containing 3 V_(H), all 27 D_(H) and 9 J_(H) human gene segmentsinserted into the proximal end of the mouse IgH locus, with aconcomitant 16.6 kb deletion of mouse genomic sequence, using about 75kb of mouse homology arms (Step A, FIG. 2A; Tables 1 and 3, 3hV_(H)).This large 144 kb insertion and accompanying 16.6 kb deletion wasperformed in a single step (Step A) that occurred with a frequency of0.2% (Table 3). Correctly targeted ES cells were scored by aloss-of-native-allele (LONA) assay (Valenzuela et al., 2003) usingprobes within and flanking the deleted mouse sequence and within theinserted human sequence, and the integrity of the large human insert wasverified using multiple probes spanning the entire insertion (FIGS. 3A,3B and 3C). Because many rounds of sequential ES cell targeting wereanticipated, targeted ES cell clones at this, and all subsequent, stepswere subjected to karyotypic analysis (supra) and only those clonesshowing normal karyotypes in at least 17 of 20 spreads were utilized forsubsequent steps.

Targeted ES cells from Step A were re-targeted with a BACvec thatproduced a 19 kb deletion at the distal end of the heavy chain locus(Step B, FIG. 2A). The Step B BACvec contained a hygromycin resistancegene (hyg) in contrast to the neomycin resistance gene (neo) containedon the BACvec of Step A. The resistance genes from the two BACvecs weredesigned such that, upon successful targeting to the same chromosome,approximately three Mb of the mouse heavy chain variable gene locuscontaining all of the mouse V_(H) gene segments other than V_(H)1-86 andall of the D_(H) gene segments other than DQ52, as well as the tworesistance genes, were flanked by IoxP sites; DQ52 and all of the mouseJ_(H) chain gene segments were deleted in Step A. ES cell clones doublytargeted on the same chromosome were identified by driving the 3hV_(H)proximal cassette to homozygosity in high G418 (Mortensen et al., 1992,Production of homozygous mutant ES cells with a single targetingconstruct, Mol Cell Biol 12:2391-2395) and following the fate of thedistal hyg cassette. Mouse segments up to four Mb in size, having beenmodified in a manner to be flanked by IoxP sites, have been successfullydeleted in ES cells by transient expression of CRE recombinase with highefficiencies (up to =11%) even in the absence of drug selection (Zhenget al., 2000, Engineering mouse chromosomes with Cre-IoxP: range,efficiency, and somatic applications, Mol Cell Biol 20:648-655). In asimilar manner, the inventors achieved a three Mb deletion in 8% of EScell clones following transient CRE expression (Step C, FIG. 2A; Table3). The deletion was scored by the LONA assay using probes at either endof the deleted mouse sequence, as well as the loss of neo and hyg andthe appearance of a PCR product across the deletion point containing thesole remaining IoxP site. Further, the deletion was confirmed byfluorescence in situ hybridization (data not shown).

The remainder of the human heavy chain variable region was added to the3hV_(H) allele in a series of 5 steps using the VELOCIGENE® geneticengineering method (Steps E-H, FIG. 2B), with each step involvingprecise insertion of up to 210 kb of human gene sequences. For eachstep, the proximal end of each new BACvec was designed to overlap themost distal human sequences of the previous step and the distal end ofeach new BACvec contained the same distal region of mouse homology asused in Step A. The BACvecs of steps D, F and H contained neo selectioncassettes, whereas those of steps E and G contained hyg selectioncassettes, thus selections were alternated between G418 and hygromycin.Targeting in Step D was assayed by the loss of the unique PCR productacross the distal IoxP site of 3hV_(H) Hybrid Allele. Targeting forSteps E through I was assayed by loss of the previous selectioncassette. In the final step (Step 1, FIG. 2B), the neo selectioncassette, flanked by Frt sites (McLeod et al., 1986, Identification ofthe crossover site during FLP-mediated recombination in theSaccharomyces cerevisiae plasmid 2 microns circle, Mol Cell Biol6:3357-3367), was removed by transient FLPe expression (Buchholz et al.,1998, Improved properties of FLP recombinase evolved by cyclingmutagenesis, Nat Biotechnol 16:657-662). The human sequences of theBACvecs for Steps D, E and G were derived from two parental human BACseach, whereas those from Steps F and H were from single BACs. Retentionof human sequences was confirmed at every step using multiple probesspanning the inserted human sequences (as described above, e.g., FIGS.3A, 3B and 3C). Only those clones with normal karyotype and germlinepotential were carried forward in each step. ES cells from the finalstep were still able to contribute to the germline after nine sequentialmanipulations (Table 3). Mice homozygous for each of the heavy chainalleles were viable, appeared healthy and demonstrated an essentiallywild-type humoral immune system (see Example 3).

TABLE 3 Hybrid Targeting Targeting Allele Human sequence constructefficiency % usage Total V_(H) Functional V_(H) 3hV_(H) 144 kb 240 kb0.2% 5 3 3 3hV_(H)/DC 144 kb 110 kb 0.1% 5 3 3 3hV_(H)-CRE 144 kb —   8%5 3 3 18hV_(H) 340 kb 272 kb 0.1% 25 18 12 39hV_(H) 550 kb 282 kb 0.2%60 39 25 53hV_(H) 655 kb 186 kb 0.4% 65 53 29 70hV_(H) 850 kb 238 kb0.5% 90 70 39 80hV_(H) 940 kb 124 kb 0.2% 100 80 43 80hV_(H)dNeo 940 kb— 2.6% 100 80 43

Immunoglobulin κ Light Chain Variable Gene Locus.

The κ light chain variable region was humanized in eight sequentialsteps by the direct replacement of about three Mb of mouse sequencecontaining all Vκ and Jκ gene segments with about 0.5 Mb of humansequence containing the proximal human Vκ and Jκ gene segments in amanner similar to that of the heavy chain (FIG. 1B; Tables 2 and 4).

The variable region of the human κ light chain locus contains two nearlyidentical 400 kb repeats separated by an 800 kb spacer (Weichhold etal., 1993, The human immunoglobulin kappa locus consists of two copiesthat are organized in opposite polarity, Genomics 16:503-511). Becausethe repeats are so similar, nearly all of the locus diversity can bereproduced in mice by using the proximal repeat. Further, a naturalhuman allele of the κ light chain locus missing the distal repeat hasbeen reported (Schaible et al., 1993, The immunoglobulin kappa locus:polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals,Hum Genet. 91:261-267). The inventors replaced about three Mb of mouse κlight chain variable gene sequence with about 0.5 Mb of human κ lightchain variable gene sequence to effectively replace all of the mouse Vκand Jκ gene segments with the proximal human Vκ and all of the human Jκgene segments (FIGS. 2C and 2D; Tables 2 and 4). In contrast to themethod described in Example 1 for the heavy chain locus, the entiremouse Vκ gene region, containing all Vκ and Jκ gene segments, wasdeleted in a three-step process before any human sequence was added.First, a neo cassette was introduced at the proximal end of the variableregion (Step A, FIG. 2C). Next, a hyg cassette was inserted at thedistal end of the κ locus (Step B, FIG. 2C). Recombinase recognitionsites (e.g., IoxP) were again situated within each selection cassettesuch that CRE treatment induced deletion of the remaining 3 Mb of themouse Vκ region along with both resistance genes (Step C, FIG. 2C).

A human genomic fragment of about 480 kb in size containing the entireimmunoglobulin κ light chain variable region was inserted in foursequential steps (FIG. 2D; Tables 2 and 4), with up to 150 kb of humanimmunoglobulin κ light chain sequence inserted in a single step, usingmethods similar to those employed for the heavy chain (see Example 1).The final hygromycin resistance gene was removed by transient FLPeexpression. As with the heavy chain, targeted ES cell clones wereevaluated for integrity of the entire human insert, normal karyotype andgerm-line potential after every step. Mice homozygous for each of the κlight chain alleles were generated and found to be healthy and of normalappearance.

TABLE 4 Hybrid Human Targeting % Allele sequence construct Targetingefficiency usage Total Vκ Functional Vκ Igκ-PC 0 132 kb 1.1% — — —Igκ-PC/DC 0  90 kb 0.4% — — — Igκ-CRE 0 —   1% — — — 6hVκ 110 kb 122 kb0.3% 14 6 4 16hVκ 240 kb 203 kb 0.4% 47 16 11 30hVκ 390 kb 193 kb 0.1%70 30 18 40hVκ 480 kb 185 kb 0.2% 100 40 25 40hVκdHyg 480 kb — 0.7% 10040 25

Example 2 Generation of Fully Humanized Mice by Combination of MultipleHumanized Immunoglobulin Alleles

At several points, ES cells bearing a portion of the humanimmunoglobulin heavy chain or κ light chain variable repertoires asdescribed in Example 1 were microinjected and the resulting mice bred tocreate multiple versions of VELOCIMMUNE® mice with progressively largerfractions of the human germline immunoglobulin repertoires (Table 5;FIGS. 5A and 5B). VELOCIMMUNE® 1 (V1) mice possess eighteen human V_(H)gene segments and all of the human D_(H) and J_(H) gene segmentscombined with sixteen human Vκ gene segments and all the human Jκ genesegments. VELOCIMMUNE® 2 (V2) and VELOCIMMUNE® (V3) mice have increasedvariable repertoires bearing a total of thirty-nine V_(H) and thirty Vκ,and eighty V_(H) and forty Vκ, respectively. Since the genomic regionsencoding the mouse V_(H), D_(H) and J_(H) gene segments, and Vκ and Jκgene segments, have been completely replaced, antibodies produced by allversions of VELOCIMMUNE® mice contain human variable regions linked tomouse constant regions. The mouse λ light chain loci remain intact invarious embodiments of the VELOCIMMUNE® mice and serve as a comparatorfor efficiency of expression of the various VELOCIMMUNE® κ light chainloci.

Mice doubly homozygous for both immunoglobulin heavy chain and κ lightchain humanizations were generated from a subset of the allelesdescribed in Example 1. All genotypes observed during the course ofbreeding to generate the doubly homozygous mice occurred in roughlyMendelian proportions. Male progeny homozygous for each of the humanheavy chain alleles demonstrated reduced fertility, which resulted fromloss of mouse ADAM6 activity. The mouse heavy chain variable gene locuscontains two embedded functional ADAM6 genes (ADAM6a and ADAM6b). Duringhumanization of the mouse heavy chain variable gene locus, the insertedhuman genomic sequence contained an ADAM6 pseudogene. Mouse ADAM6 may berequired for fertility, and thus lack of mouse ADAM6 genes in humanizedheavy chain variable gene loci might lead to a reduction in fertilitynotwithstanding the presence of the human pseudogene. Examples 7-11describe the reengineering of mouse ADAM6 genes into a humanized heavychain variable gene locus, and restoration of wild-type level fertilityin mice with a humanized heavy chain immunoglobulin locus.

TABLE 5 Version of Heavy Chain κ Light Chain VELOCIMMUNE ® Human MouseV_(H) Allele 5′ V_(H) gene Human Vκ Allele 5′ Vκ gene V1 18 18hV_(H)V_(H)1-18 16 16hVκ Vκ1-16 V2 39 39hV_(H) V_(H)4-39 30 30hVκ Vκ2-29 V3 8080hV_(H) V_(H)3-74 40 40hVκ Vκ2-40

Example 3 Lymphocyte Populations in Mice with Humanized ImmunoglobulinGenes

Mature B cell populations in the three different versions ofVELOCIMMUNE® mice were evaluated by flow cytometry.

Briefly, cell suspensions from bone marrow, spleen and thymus were madeusing standard methods. Cells were resuspended at 5×10⁵ cells/mL in BDPharmingen FACS staining buffer, blocked with anti-mouse CD16/32 (BDPharmingen), stained with the appropriate cocktail of antibodies andfixed with BD CYTOFIX™ all according to the manufacturer's instructions.Final cell pellets were resuspended in 0.5 mL staining buffer andanalyzed using a BD FACSCALIBUR™ and BD CELLQUEST PRO™ software. Allantibodies (BD Pharmingen) were prepared in a mass dilution/cocktail andadded to a final concentration of 0.5 mg/10⁵ cells.

Antibody cocktails for bone marrow (A-D) staining were as follows: A:anti-mouse IgM^(b)-FITC, anti-mouse IgM^(a)-PE, anti-mouseCD45R(B220)-APC; B: anti-mouse CD43(S7)-PE, anti-mouse CD45R(B220)-APC;C: anti-mouse CD24(HSA)-PE; anti-mouse CD45R(B220)-APC; D: anti-mouseBP-1-PE, anti-mouse CD45R(B220)-APC.

Antibody cocktails for spleen and inguinal lymph node (E-H) stainingwere as follows: E: anti-mouse IgM^(b)-FITC, anti-mouse IgM^(a)-PE,anti-mouse CD45R(B220)-APC; F: anti-mouse Ig, λ1, λ2, λ3 LightChain-FITC, anti mouse Igκ Light Chain-PE, anti-mouse CD45R(B220)-APC;G: anti-mouse Ly6G/C-FITC, anti-mouse CD49b(DX5)-PE, anti-mouseCD11b-APC; H: anti-mouse CD4(L3T4)-FITC, anti-mouse CD45R(B220)-PE,anti-mouse CD8a-APC. Results are shown in FIG. 6.

Lymphocytes isolated from spleen or lymph node of homozygousVELOCIMMUNE® mice were stained for surface expression of the markersB220 and IgM and analyzed using flow cytometry (FIG. 6). The sizes ofthe B220⁺ IgM⁺ mature B cell populations in all versions of VELOCIMMUNE®mice tested were virtually identical to those of wild type mice,regardless of the number of V_(H) gene segments they contained. Inaddition, mice containing homozygous hybrid humanized immunoglobulinheavy chain loci, even those with only 3 V_(H) gene segments but normalmouse immunoglobulin κ light chain loci or mice containing homozygoushybrid humanized κ light chain loci with normal mouse immunoglobulinheavy chain loci, also had normal numbers of B220⁺ IgM⁺ cells in theirperipheral compartments (not shown). These results indicate thatchimeric loci with human variable gene segments and mouse constantregions can fully populate the mature B cell compartment. Further, thenumber of variable gene segments at either the heavy chain or K lightchain loci, and thus the theoretical diversity of the antibodyrepertoire, does not correlate with the ability to generate wild typepopulations of mature B cells. In contrast, mice with randomlyintegrated fully-human immunoglobulin transgenes and inactivated mouseimmunoglobulin loci have reduced numbers of B cells in thesecompartments, with the severity of the deficit depending on the numberof variable gene segments included in the transgene (Green andJakobovits, 1998, Regulation of B cell development by variable genecomplexity in mice reconstituted with human immunoglobulin yeastartificial chromosomes, J Exp Med 188:483-495). This demonstrates thatthe “in situ genetic humanization” strategy results in a fundamentallydifferent functional outcome than the randomly integrated transgenesachieved in the “knockout-plus-transgenic” approach.

Allelic Exclusion and Locus Choice.

The ability to maintain allelic exclusion was examined in miceheterozygous for different versions of the humanized immunoglobulinheavy chain locus.

The humanization of the immunoglobulin loci was carried out in an F1 ESline (F1H4, Valenzuela et al., 2003), derived from 12956/SvEvTac andC57BL/6NTac heterozygous embryos. The human heavy chain germlinevariable gene sequences are targeted to the 129S6 allele, which carriesthe IgM^(a) haplotype, whereas the unmodified mouse C576BL/6N allelebears the IgM^(b) haplotype. These allelic forms of IgM can bedistinguished by flow cytometry using antibodies specific to thepolymorphisms found in the IgM^(a) or IgM^(b) alleles. As shown in FIG.6 (bottom row), the B cells identified in mice heterozygous for eachversion of the humanized heavy chain locus only express a single allele,either IgM^(a) (the humanized allele) or IgM^(b) (the wild type allele).This demonstrates that the mechanisms involved in allelic exclusion areintact in VELOCIMMUNE® mice. In addition, the relative number of B cellspositive for the humanized allele (IgM^(a)) is roughly proportional tothe number of V_(H) gene segments present. The humanized immunoglobulinlocus is expressed in approximately 30% of the B cells in VELOCIMMUNE® 1heterozygote mice, which have 18 human V_(H) gene segments, and in 50%of the B cells in VELOCIMMUNE® 2 and 3 (not shown) heterozygote mice,with 39 and 80 human V_(H) gene segments, respectively. Notably, theratio of cells expressing the humanized versus wild type mouse allele(0.5 for VELOCIMMUNE® 1 mice and 0.9 for VELOCIMMUNE® 2 mice) is greaterthan the ratio of the number of variable gene segments contained in thehumanized versus wild type loci (0.2 for VELOCIMMUNE® 1 mice and 0.4 forVELOCIMMUNE® 2 mice). This may indicate that the probability of allelechoice is intermediate between a random choice of one or the otherchromosome and a random choice of any particular V segment RSS. Further,there may be a fraction of B-cells, but not all, in which one allelebecomes accessible for recombination, completes the process and shutsdown recombination before the other allele becomes accessible. Inaddition, the even distribution of cells that have surface IgM (sIgM)derived from either the hybrid humanized heavy chain locus or the wildtype mouse heavy chain locus is evidence that the hybrid locus isoperating at a normal level. In contrast, randomly integrated humanimmunoglobulin transgenes compete poorly with wild type mouseimmunoglobulin loci (Bruggemann et al., 1989, A repertoire of monoclonalantibodies with human heavy chains from transgenic mice, PNAS86:6709-6713; Green et al., 1994; Tuaillon et al., 1993, Humanimmunoglobulin heavy-chain minilocus recombination in transgenic mice:gene-segment use in mu and gamma transcripts, PNAS USA 90:3720-3724).This further demonstrates the immunoglobulins produced by VELOCIMMUNE®mice are functionally different than those produced by randomlyintegrated transgenes in mice made by “knockout-plus-transgenic”approaches.

Polymorphisms of the Cκ regions are not available in 12956 or C57BL/6Nto examine allelic exclusion of humanized versus non-humanized κ lightchain loci. However, VELOCIMMUNE® mice all possess wild type mouse λlight chain loci, therefore, it is possible to observe whetherrearrangement and expression of humanized κ light chain loci can preventmouse λlight chain expression. The ratio of the number of cellsexpressing the humanized κ light chain relative to the number of cellsexpressing mouse λlight chain was relatively unchanged in VELOCIMMUNE®mice compared with wild type mice, regardless of the number of human Vκgene segments inserted at the κ light chain locus (FIG. 6, third rowfrom top). In addition there was no increase in the number of doublepositive (κ plus λ) cells, indicating that productive recombination atthe hybrid κ light chain loci results in appropriate suppression ofrecombination of the mouse λ light chain loci. In contrast, micecontaining randomly integrated κ light chain transgenes with inactivatedmouse κ light chain loci—but wild type mouse λlight chain loci—exhibitdramatically increased λ/κ ratios (Jakobovits, 1998), implying that theintroduced κ light chain transgenes do not function well in such mice.This further demonstrates the different functional outcome observed inimmunoglobulins made by VELOCIMMUNE® mice as compared to those made by“knockout-plus-transgenic” mice.

B Cell Development.

Because the mature B cell populations in VELOCIMMUNE® mice resemblethose of wild type mice (described above), it is possible that defectsin early B cell differentiation are compensated for by the expansion ofmature B cell populations. The various stages of B cell differentiationwere examined by analysis of B cell populations using flow cytometry.Table 6 sets forth the ratio of the fraction of cells in each B celllineage defined by FACs, using specific cell surface markers, inVELOCIMMUNE®, mice compared to wild type littermates.

Early B cell development occurs in the bone marrow, and different stagesof B cell differentiation are characterized by changes in the types andamounts of cell surface marker expression. These differences in surfaceexpression correlate with the molecular changes occurring at theimmunoglobulin loci inside the cell. The pro-B to pre-B cell transitionrequires the successful rearrangement and expression of functional heavychain protein, while transition from the pre-B to mature B stage isgoverned by the correct rearrangement and expression of a κ or λ lightchain. Thus, inefficient transition between stages of B celldifferentiation can be detected by changes in the relative populationsof B cells at a given stage.

TABLE 6 Bone Marrow Spleen Emerging Version of pro-B pre-B ImmatureMature B220^(hi) Mature VELOCIMMUNE ® CD43^(hi) CD24^(hi) B220^(lo)B220^(hi) IgM⁺ B220hi Mice B220^(lo) B220^(lo) IgM⁺ IgM⁺ IgD⁺ IgM⁺ V11.1 1.0 0.9 1.0 1.1 1.0 V2 1.0 1.0 1.0 1.0 1.0 1.0 V3 1.0 1.0 1.1 1.01.0 1.1

No major defects were observed in B cell differentiation in any of theVELOCIMMUNE® mice. The introduction of human heavy chain gene segmentsdoes not appear to affect the pro-B to pre-B transition, andintroduction of human κ light chain gene segments does not affect thepre-B to B transition in VELOCIMMUNE® mice. This demonstrates that“reverse chimeric” immunoglobulin molecules possessing human variableregions and mouse constants function normally in the context of B cellsignaling and co-receptor molecules leading to appropriate B celldifferentiation in a mouse environment. In contrast, the balance betweenthe different populations during B cell differentiation are perturbed tovarying extents in mice that contain randomly integrated immunoglobulintransgenes and inactivated endogenous heavy chain or κ light chain loci(Green and Jakobovits, 1998).

Example 4 Variable Gene Repertoire in Humanized Immunoglobulin Mice

Usage of human variable gene segments in the humanized antibodyrepertoire of VELOCIMMUNE® mice was analyzed by reversetranscriptase-polymerase chain reaction (RT-PCR) of human variableregions from multiple sources including splenocytes and hybridoma cells.Variable region sequence, gene segment usage, somatic hypermutation, andjunctional diversity of rearranged variable region gene segments weredetermined.

Briefly, total RNA was extracted from 1×10⁷-2×10⁷ splenocytes or about10⁴-10⁵ hybridoma cells using TRIZOL™ (Invitrogen) or Qiagen RNEASY™Mini Kit (Qiagen) and primed with mouse constant region specific primersusing the SUPERSCRIPT™ III One-Step RT-PCR system (Invitrogen).Reactions were carried out with 2-5 μL of RNA from each sample using theaforementioned 3′ constant specific primers paired with pooled leaderprimers for each family of human variable regions for both the heavychain and κ light chain, separately. Volumes of reagents and primers,and RT-PCR/PCR conditions were performed according to the manufacturer'sinstructions. Primers sequences were based upon multiple sources (Wangand Stollar, 2000, Human immunoglobulin variable region gene analysis bysingle cell RT-PCR, J Immunol Methods 244:217-225; Ig-primer sets,Novagen). Where appropriate, nested secondary PCR reactions were carriedout with pooled family-specific framework primers and the same mouse 3′immunoglobulin constant-specific primer used in the primary reaction.Aliquots (5 μL) from each reaction were analyzed by agaroseelectrophoresis and reaction products were purified from agarose using aMONTAGE™ Gel Extraction Kit (Millipore). Purified products were clonedusing the TOPO™ TA Cloning System (Invitrogen) and transformed intoDH10β E. coli cells by electroporation. Individual clones were selectedfrom each transformation reaction and grown in 2 mL LB broth cultureswith antibiotic selection overnight at 37° C. Plasmid DNA was purifiedfrom bacterial cultures by a kit-based approach (Qiagen).

Immunoglobulin Variable Gene Usage.

Plasmid DNA of both heavy chain and K light chain clones were sequencedwith either T7 or M13 reverse primers on the ABI 3100 Genetic Analyzer(Applied Biosystems). Raw sequence data were imported into SEQUENCHER™(v4.5, Gene Codes). Each sequence was assembled into contigs and alignedto human immunoglobulin sequences using IMGT V-Quest (Brochet et al.,2008, IMGT/V-QUEST: the highly customized and integrated system for IGand TR standardized V-J and V-D-J sequence analysis, Nucleic Acids Res36:Vκ503-508) search function to identify human V_(H), D_(H), J_(H) andVκ, Jκ segment usage. Sequences were compared to germline sequences forsomatic hypermutation and recombination junction analysis.

Mice were generated from ES cells containing the initial heavy chainmodification (3hV_(H)-CRE Hybrid Allele, bottom of FIG. 2A) by RAGcomplementation (Chen et al., 1993, RAG-2-deficient blastocystcomplementation: an assay of gene function in lymphocyte development,PNAS USA 90:4528-4532), and cDNA was prepared from splenocyte RNA. ThecDNA was amplified using primer sets (described above) specific for thepredicted chimeric heavy chain mRNA that would arise by V(D)Jrecombination within the inserted human gene segments and subsequentsplicing to either mouse IgM or IgG constant domains. Sequences derivedfrom these cDNA clones (not shown) demonstrated that proper V(D)Jrecombination had occurred within the human variable gene sequences,that the rearranged human V(D)J gene segments were properly splicedin-frame to mouse constant domains, and that class-switch recombinationhad occurred. Further sequence analysis of mRNA products of subsequenthybrid immunoglobulin loci was performed.

In a similar experiment, B cells from non-immunized wild type andVELOCIMMUNE® mice were separated by flow cytometry based upon surfaceexpression of B220 and IgM or IgG. The B220⁺ IgM⁺ or surface IgG⁺(sIgG⁺) cells were pooled and V_(H) and Vκ sequences were obtainedfollowing RT-PCR amplification and cloning (described above).Representative gene usage in a set of RT-PCR amplified cDNAs fromunimmunized VELOCIMMUNE® 1 mice (Table 7) and VELOCIMMUNE® 3 mice (Table8) was recorded (*defective RSS; †missing or pseudogene). Asterisk: genesegments with defective RSS. †: gene segment is missing or pseudogene.

TABLE 7 Observed V_(H) 1-18 3 1-17P 0 3-16* 0 3-15 13 3-13 9 3-11 6 3-98 1-8 6 3-7 2 2-5 2 1-3 0 1-2 11 6-1 5 J_(H) 1 2 2 1 3 8 4 33 5 5 6 16D_(H) 1-1 1 2-2 2 3-3 4 4-4 0 5-5 0 5-18 4 6-6 5 1-7 7 2-8 0 3-9 4 3-102 4-11 1 5-12 1 6-13 3 1-14 0 2-15 0 3-16 1 4-17 0 6-19 2 1-20 2 2-21 13-22 0 4-23 2 5-24 1 6-25 1 1-26 6 7-27 10 Vκ 1-16 2 3-15 1 1-12 5 3-111 1-9 5 1-8 2 3-7* 0 1-6 5 1-5 8 5-2 6 4-1 8 Jκ 1 12 2 10 3 5 4 10 5 0

TABLE 8 Observed V_(H) 7-81† 0 3-74† 0 3-73 1 3-72 2 2-70 2 1-69 3 3-661 3-64 1 4-61 1 4-59 10 1-58 0 3-53 0 5-51 5 3-49 2 3-48 7 1-46 1 1-45 03-43 10 4-39 4 3-38* 0 3-35* 0 4-34 8 3-33 14 4-31 4 3-30 13 4-28 0 2-260 1-24 3 3-23 18 3-21 0 3-20 0 1-18 4 1-17P 1 3-16* 0 3-15 13 3-13 63-11 5 3-9 31 1-8 7 3-7 11 2-5 1 1-3 0 1-2 6 6-1 9 D_(H) 1-1 7 2-2 8 3-39 4-4 4 5-5 6 5-18 6 6-6 29 1-7 30 2-8 4 3-9 8 3-10 10 4-11 4 5-12 56-13 17 1-14 2 2-15 3 3-16 4 4-17 3 6-19 8 1-20 3 2-21 1 3-22 5 4-23 25-24 2 6-25 2 1-26 17 7-27 7 J_(H) 1 2 2 8 3 26 4 95 5 11 6 58 Vκ 2-40 11-39 34 1-37 2 1-33 35 2-30 8 2-29 2 2-28 7 1-27 5 2-24 7 6-21* 3 3-2010 1-17 13 1-16 10 3-15 13 1-12 13 3-11 13 1-9 11 1-8 1 3-7* 0 1-6 6 1-57 5-2 0 4-1 21 Jκ 1 50 2 37 3 28 4 64 5 22

As shown in Tables 7 and 8, nearly all of the functional human V_(H),D_(H), J_(H), Vκ and Jκ gene segments are utilized. Of the functionalvariable gene segments described but not detected in the VELOCIMMUNE®mice of this experiment, several have been reported to possess defectiverecombination signal sequences (RSS) and, thus, would not be expected tobe expressed (Feeney, 2000, Factors that influence formation of B cellrepertoire, Immunol Res 21:195-202). Analysis of several other sets ofimmunoglobulin sequences from various VELOCIMMUNE® mice, isolated fromboth naïve and immunized repertoires, has shown usage of these genesegments, albeit at lower frequencies (data not shown). Aggregate geneusage data has shown that all functional human V_(H), D_(H), J_(H), Vκ,and Jκ gene segments contained in VELOCIMMUNE® mice have been observedin various naïve and immunized repertoires (data not shown). Althoughthe human V_(H)7-81 gene segment has been identified in the analysis ofhuman heavy chain locus sequences (Matsuda et al., 1998, The completenucleotide sequence of the human immunoglobulin heavy chain variableregion locus, J Exp Med 188:2151-2162), it is not present in theVELOCIMMUNE® mice as confirmed by re-sequencing of the entireVELOCIMMUNE® 3 mouse genome.

Sequences of heavy and light chains of antibodies are known to showexceptional variability, especially in short polypeptide segments withinthe rearranged variable domain. These regions, known as hypervariableregions or complementary determining regions (CDRs), create the bindingsite for antigen in the structure of the antibody molecule. Theintervening polypeptide sequences are called framework regions (FRs).There are three CDRs (CDR1, CDR2, CDR3) and 4 FRs (FR1, FR2, FR3, FR4)in both heavy and light chains. One CDR, CDR3, is unique in that thisCDR is created by recombination of both the V_(H), D_(H) and J_(H) andVκ and Jκ gene segments and generates a significant amount of repertoirediversity before antigen is encountered. This joining is imprecise dueto both nucleotide deletions via exonuclease activity and non-templateencoded additions via terminal deoxynucleotidyl transferase (TdT) and,thus, allows for novel sequences to result from the recombinationprocess. Although FRs can show substantial somatic mutation due to thehigh mutability of the variable region as a whole, variability is not,however, distributed evenly across the variable region. CDRs areconcentrated and localized regions of high variability in the surface ofthe antibody molecule that allow for antigen binding. Heavy chain andlight chain sequences of selected antibodies from VELOCIMMUNE® micearound the CDR3 junction demonstrating junctional diversity are shown inFIGS. 7A and 7B, respectively.

As shown in FIG. 7A, non-template encoded nucleotide additions(N-additions) are observed at both the V_(H)-D_(H) and D_(H)-J_(H) jointin antibodies from VELOCIMMUNE® mice, indicating proper function of TdTwith the human segments. The endpoints of the V_(H), D_(H) and J_(H)segments relative to their germline counterparts indicate thatexonuclease activity has also occurred. Unlike the heavy chain locus,the human κ light chain rearrangements exhibit little or no TdTadditions at CDR3, which is formed by the recombination of the Vκ and Jκsegments (FIG. 7B). This is expected due to the lack of TdT expressionin mice during light chain rearrangements at the pre-B to B celltransition. The diversity observed in the CDR3 of rearranged human Vκregions is introduced predominantly through exonuclease activity duringthe recombination event.

Somatic Hypermutation.

Additional diversity is added to the variable regions of rearrangedimmunoglobulin genes during the germinal center reaction by a processtermed somatic hypermutation. B cells expressing somatically mutatedvariable regions compete with other B cells for access to antigenpresented by the follicular dendritic cells. Those B cells with higheraffinity for the antigen will further expand and undergo class switchingbefore exiting to the periphery. Thus, B cells expressing switchedisotypes typically have encountered antigen and undergone germinalcenter reactions and will have increased numbers of mutations relativeto naïve B cells. Further, variable region sequences from predominantlynaïve sIgM⁺ B cells would be expected to have relatively fewer mutationsthan variable sequences from sIgG⁺ B cells which have undergone antigenselection.

Sequences from random V_(H) or Vκ clones from sIgM⁺ or sIgG⁺ B cellsfrom non-immunized VELOCIMMUNE® mice or sIgG⁺ B cells from immunizedmice were compared with their germline variable gene segments andchanges relative to the germline sequence annotated. The resultingnucleotide sequences were translated in silico and mutations leading toamino acid changes also annotated. The data were collated from all thevariable regions and the percent change at a given position wascalculated (FIG. 8).

As shown in FIG. 8, human heavy chain variable regions derived fromsIgG⁺ B cells from non-immunized VELOCIMMUNE® mice exhibit many morenucleotides relative to sIgM⁺ B cells from the same splenocyte pools,and heavy chain variable regions derived from immunized mice exhibiteven more changes. The number of changes is increased in thecomplementarity-determining regions (CDRs) relative to the frameworkregions, indicating antigen selection. The corresponding amino acidsequences from the human heavy chain variable regions also exhibitsignificantly higher numbers of mutations in IgG versus IgM and evenmore in immunized IgG. These mutations again appear to be more frequentin the CDRs compared with the framework sequences, suggesting that theantibodies were antigen-selected in vivo. A similar increase in thenumber the nucleotide and amino acid mutations are seen in the Vκsequences derived from IgG⁺ B cells from immunized mice.

The gene usage and somatic hypermutation frequency observed inVELOCIMMUNE® mice demonstrate that essentially all gene segments presentare capable of rearrangement to form fully functionally reverse chimericantibodies in these mice. Further, VELOCIMMUNE® antibodies fullyparticipate within the mouse immune system to undergo affinity selectionand maturation to create fully mature human antibodies that caneffectively neutralize their target antigen. VELOCIMMUNE® mice are ableto mount robust immune responses to multiple classes of antigens thatresult in usage of a wide range of human antibodies that are both highaffinity and suitable for therapeutic use (data not shown).

Example 5 Analysis of Lymphoid Structure and Serum Isotypes

The gross structures of spleen, inguinal lymph nodes, Peyer's patchesand thymus of tissue samples from wild type or VELOCIMMUNE® mice stainedwith H&E were examined by light microscopy. The levels of immunoglobulinisotypes in serum collected from wild type and VELOCIMMUNE® mice wereanalyzed using LUMINEX™ technology.

Lymphoid Organ Structure.

The structure and function of the lymphoid tissues are in part dependentupon the proper development of hematopoietic cells. A defect in B celldevelopment or function may be exhibited as an alteration in thestructure of the lymphoid tissues. Upon analysis of stained tissuesections, no significant difference in appearance of secondary lymphoidorgans between wild type and VELOCIMMUNE® mice was identified (data notshown).

Serum Immunoglobulin Levels.

The level of expression of each isotype is similar in wild type andVELOCIMMUNE® mice (FIGS. 9A, 9B and 9C). This demonstrates thathumanization of the variable gene segments had no apparent adverseeffect upon class switching or immunoglobulin expression and secretionand therefore apparently maintain all the endogenous mouse sequencesnecessary for these functions.

Example 6 Immunization and Antibody Production in HumanizedImmunoglobulin Mice

Different versions of VELOCIMMUNE® mice were immunized with antigen toexamine the humoral response to foreign antigen challenge.

Immunization and Hybridoma Development.

VELOCIMMUNE® and wild-type mice can be immunized with an antigen in theform of protein, DNA, a combination of DNA and protein, or cellsexpressing the antigen. Animals are typically boosted every three weeksfor a total of two to three times. Following each antigen boost, serumsamples from each animal are collected and analyzed for antigen-specificantibody responses by serum titer determination. Prior to fusion, micereceived a final pre-fusion boost of 5 μg protein or DNA, as desired,via intra-peritoneal and/or intravenous injections. Splenocytes areharvested and fused to Ag8.653 myeloma cells in an electrofusion chamberaccording to the manufacture's suggested protocol (Cyto Pulse SciencesInc., Glen Burnie, Md.). Ten days after culture, hybridomas are screenedfor antigen specificity using an ELISA assay (Harlow and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York).Alternatively, antigen specific B cells are isolated directly fromimmunized VELOCIMMUNE® mice and screened using standard techniques,including those described here, to obtain human antibodies specific foran antigen of interest (e.g., see US 2007/0280945A1, herein incorporatedby reference in its entirety).

Serum Titer Determination.

To monitor animal anti-antigen serum response, serum samples arecollected about 10 days after each boost and the titers are determinedusing antigen specific ELISA. Briefly, Nunc MAXISORP™ 96 well plates arecoated with 2 μg/mL antigen overnight at 4° C. and blocked with bovineserum albumin (Sigma, St. Louis, Mo.). Serum samples in a serial 3 folddilutions are allowed to bind to the plates for one hour at roomtemperature. The plates are then washed with PBS containing 0.05%Tween-20 and the bound IgG are detected using HRP-conjugated goatanti-mouse Fc (Jackson Immuno Research Laboratories, Inc., West Grove,Pa.) for total IgG titer, or biotin-labeled isotype specific or lightchain specific polyclonal antibodies (Southern Biotech Inc.) for isotypespecific titers, respectively. For biotin-labeled antibodies, followingplate wash, HRP-conjugated streptavidin (Pierce, Rockford, Ill.) isadded. All plates are developed using colorimetric substrates such as BDOPTEIA™ (BD Biosciences Pharmingen, San Diego, Calif.). After thereaction is stopped with 1 M phosphoric acid, optical absorptions at 450nm are recorded and the data are analyzed using PRISM™ software fromGraph Pad. Dilutions required to obtain two-fold of background signalare defined as titer.

In one experiment, VELOCIMMUNE® mice were immunized with humaninterleukin-6 receptor (hIL-6R). A representative set of serum titersfor VELOCIMMUNE® and wild type mice immunized with hIL-6R is shown inFIGS. 10A and 10B.

VELOCIMMUNE® and wild-type mice mounted strong responses towards IL-6Rwith similar titer ranges (FIG. 10A). Several mice from the VELOCIMMUNE®and wild-type cohorts reached a maximal response after a single antigenboost. These results indicate that the immune response strength andkinetics to this antigen were similar in the VELOCIMMUNE® and wild typemice. These antigen-specific antibody responses were further analyzed toexamine the particular isotypes of the antigen-specific antibodies foundin the sera. Both VELOCIMMUNE® and wild type groups predominantlyelicited an IgG1 response (FIG. 10B), suggesting that class switchingduring the humoral response is similar in mice of each type.

Affinity Determination of Antibody Binding to Antigen in Solution.

An ELISA-based solution competition assay is typically designed todetermine antibody-binding affinity to the antigen.

Briefly, antibodies in conditioned medium are premixed with serialdilutions of antigen protein ranging from 0 to 10 mg/mL. The solutionsof the antibody and antigen mixture are then incubated for two to fourhours at room temperature to reach binding equilibria. The amounts offree antibody in the mixtures are then measured using a quantitativesandwich ELISA. Ninety-six well MAXISORB™ plates (VWR, West Chester,Pa.) are coated with 1 μg/mL antigen protein in PBS solution overnightat 4° C. followed by BSA nonspecific blocking. The antibody-antigenmixture solutions are then transferred to these plates followed byone-hour incubation. The plates are then washed with washing buffer andthe plate-bound antibodies were detected with an HRP-conjugated goatanti-mouse IgG polyclonal antibody reagent (Jackson Immuno Research Lab)and developed using colorimetric substrates such as BD OPTEIA™ (BDBiosciences Pharmingen, San Diego, Calif.). After the reaction isstopped with 1 M phosphoric acid, optical absorptions at 450 nm arerecorded and the data are analyzed using PRISM™ software from Graph Pad.The dependency of the signals on the concentrations of antigen insolution are analyzed with a 4 parameter fit analysis and reported asIC₅₀, the antigen concentration required to achieve 50% reduction of thesignal from the antibody samples without the presence of antigen insolution.

In one experiment, VELOCIMMUNE® mice were immunized with hIL-6R (asdescribed above). FIGS. 11A and 11B show a representative set ofaffinity measurements for anti-hIL6R antibodies from VELOCIMMUNE® andwild-type mice.

After immunized mice receive a third antigen boost, serum titers aredetermined using an ELISA assay. Splenocytes are isolated from selectedwild type and VELOCIMMUNE® mouse cohorts and fused with Ag8.653 myelomacells to form hybridomas and grown under selection (as described above).Out of a total of 671 anti-IL-6R hybridomas produced, 236 were found toexpress antigen-specific antibodies. Media harvested from antigenpositive wells was used to determine the antibody affinity of binding toantigen using a solution competition ELISA. Antibodies derived fromVELOCIMMUNE® mice exhibit a wide range of affinity in binding to antigenin solution (FIG. 11A). Furthermore, 49 out of 236 anti-IL-6R hybridomaswere found to block IL-6 from binding to the receptor in an in vitrobioassay (data not shown). Further, these 49 anti-IL-6R blockingantibodies exhibited a range of high solution affinities similar to thatof blocking antibodies derived from the parallel immunization of wildtype mice (FIG. 11B).

Example 7 Construction of a Mouse ADAM6 Targeting Vector

Due to replacement of mouse immunoglobulin heavy chain variable geneloci with human immunoglobulin heavy chain variable gene loci, earlyversions of VELOCIMMUNE® mice lack expression of mouse ADAM6 genes. Inparticular, male VELOCIMMUNE® mice demonstrate a reduction in fertility.Thus, the ability to express ADAM6 was reengineered into VELOCIMMUNE®mice to rescue the fertility defect.

A targeting vector for insertion of mouse ADAM6a and ADAM6b genes into ahumanized heavy chain locus was constructed using VELOCIGENE® geneticengineering technology (supra) to modify a Bacterial ArtificialChromosome (BAC) 929d24, which was obtained from Dr. Frederick Alt(Harvard University). 929d24 BAC DNA was engineered to contain genomicfragments containing the mouse ADAM6a and ADAM6b genes and a hygromycincassette for targeted deletion of a human ADAM6 pseudogene (hADAM64))located between human V_(H)1-2 and V_(H)6-1 gene segments of a humanizedheavy chain locus (FIG. 12).

First, a genomic fragment containing the mouse ADAM6b gene, ˜800 bp ofupstream (5′) sequence and ˜4800 bp of downstream (3′) sequence wassubcloned from the 929d24 BAC clone. A second genomic fragmentcontaining the mouse ADAM6a gene, ˜300 bp of upstream (5′) sequence and˜3400 bp of downstream (3′) sequence, was separately subcloned from the929d24 BAC clone. The two genomic fragments containing the mouse ADAM6band ADAM6a genes were ligated to a hygromycin cassette flanked by Frtrecombination sites to create the targeting vector (Mouse ADAM6Targeting Vector, FIG. 12; SEQ ID NO:3). Different restriction enzymesites were engineered onto the 5′ end of the targeting vector followingthe mouse ADAM6b gene and onto the 3′ end following the mouse ADAM6agene (bottom of FIG. 12) for ligation into the humanized heavy chainlocus.

A separate modification was made to a BAC clone containing a replacementof mouse heavy chain variable gene loci with human heavy chain variablegene loci, including the human ADAM6 pseudogene (hADAM6Ψ) locatedbetween the human V_(H)1-2 and V_(H)6-1 gene segments of the humanizedlocus for the subsequent ligation of the mouse ADAM6 targeting vector(FIG. 13).

Briefly, a neomycin cassette flanked by IoxP recombination sites wasengineered to contain homology arms containing human genomic sequence atpositions 3′ of the human V_(H)1-2 gene segment (5′ with respect tohADAM6Ψ) and 5′ of human V_(H)6-1 gene segment (3′ with respect tohADAM6Ψ; see middle of FIG. 13). The location of the insertion site ofthis targeting construct was about 1.3 kb 5′ and ˜350 bp 3′ of the humanADAM6 pseudogene. The targeting construct also included the samerestriction sites as the mouse ADAM6 targeting vector to allow forsubsequent BAC ligation between the modified BAC clone containing thedeletion of the human ADAM6 pseudogene and the mouse ADAM6 targetingvector.

Following digestion of BAC DNA derived from both constructs, the genomicfragments were ligated together to construct an engineered BAC clonecontaining a humanized heavy chain locus containing an ectopicallyplaced genomic sequence comprising mouse ADAM6a and ADAM6b nucleotidesequences. The final targeting construct for the deletion of a humanADAM6 gene within a humanized heavy chain locus and insertion of mouseADAM6a and ADAM6b sequences in ES cells contained, from 5′ to 3′, a 5′genomic fragment containing ˜13 kb of human genomic sequence 3′ of thehuman V_(H)1-2 gene segment, ˜800 bp of mouse genomic sequencedownstream of the mouse ADAM6b gene, the mouse ADAM6b gene, ˜4800 bp ofgenomic sequence upstream of the mouse ADAM6b gene, a 5′ Frt site, ahygromycin cassette, a 3′ Frt site, ˜300 bp of mouse genomic sequencedownstream of the mouse ADAM6a gene, the mouse ADAM6a gene, ˜3400 bp ofmouse genomic sequence upstream of the mouse ADAM6a gene, and a 3′genomic fragment containing ˜30 kb of human genomic sequence 5′ of thehuman V_(H)6-1 gene segment (bottom of FIG. 13).

The engineered BAC clone (described above) was used to electroporatemouse ES cells that contained a humanized heavy chain locus to createdmodified ES cells comprising a mouse genomic sequence ectopically placedthat comprises mouse ADAM6a and ADAM6b sequences within a humanizedheavy chain locus. Positive ES cells containing the ectopic mousegenomic fragment within the humanized heavy chain locus were identifiedby a quantitative PCR assay using TAQMAN™ probes (Lie and Petropoulos,1998, Advances in quantitative PCR technology: 5′ nuclease assays, CurrOpin Biotechnol 9(1):43-48). The upstream and downstream regions outsideof the modified portion of the humanized heavy chain locus wereconfirmed by PCR using primers and probes located within the modifiedregion to confirm the presence of the ectopic mouse genomic sequencewithin the humanized heavy chain locus as well as the hygromycincassette. The nucleotide sequence across the upstream insertion pointincluded the following, which indicates human heavy chain genomicsequence upstream of the insertion point and an I-Ceul restriction site(contained within the parentheses below) linked contiguously to mousegenomic sequence present at the insertion point: (CCAGCTTCAT TAGTAATCGTTCATCTGTGG TAAAAAGGCA GGATTTGAAG CGATGGAAGA TGGGAGTACG GGGCGTTGGAAGACAAAGTG CCACACAGCG CAGCCTTCGT CTAGACCCCC GGGCTAACTA TAACGGTCCTAAGGTAGCGA G) GGGATGACAG ATTCTCTGTT CAGTGCACTC AGGGTCTGCC TCCACGAGAATCACCATGCC CTTTCTCAAG ACTGTGTTCT GTGCAGTGCC CTGTCAGTGG (SEQ ID NO:4).The nucleotide sequence across the downstream insertion point at the 3′end of the targeted region included the following, which indicates mousegenomic sequence and a PI-Scel restriction site (contained within theparentheses below) linked contiguously with human heavy chain genomicsequence downstream of the insertion point: (AGGGGTCGAG GGGGAATTTTACAAAGAACA AAGAAGCGGG CATCTGCTGA CATGAGGGCC GAAGTCAGGC TCCAGGCAGCGGGAGCTCCA CCGCGGTGGC GCCATTTCAT TACCTCTTTC TCCGCACCCG ACATAGATAAAGCTT)ATCCCCCACC AAGCAAATCC CCCTACCTGG GGCCGAGCTT CCCGTATGTG GGAAAATGAATCCCTGAGGT CGATTGCTGC ATGCAATGAA ATTCAACTAG (SEQ ID NO:5).

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® mouseengineering method (see, e.g., U.S. Pat. Nos. 7,6598,442, 7,576,259,7,294,754). Mice bearing a humanized heavy chain locus containing anectopic mouse genomic sequence comprising mouse ADAM6a and ADAM6bsequences were identified by genotyping using a modification of alleleassay (Valenzuela et al., 2003) that detected the presence of the mouseADAM6a and ADAM6b genes within the humanized heavy chain locus.

Mice bearing a humanized heavy chain locus that contains mouse ADAM6aand ADAM6b genes are bred to a FLPe deleter mouse strain (see, e.g.,Rodriguez et al., 2000, High-efficiency deleter mice show that FLPe isan alternative to Cre-IoxP. Nature Genetics 25:139-140) in order toremove any Frt'ed hygromycin cassette introduced by the targeting vectorthat is not removed, e.g., at the ES cell stage or in the embryo.Optionally, the hygromycin cassette is retained in the mice.

Pups are genotyped and a pup heterozygous for a humanized heavy chainlocus containing an ectopic mouse genomic fragment that comprises mouseADAM6a and ADAM6b sequences is selected for characterizing mouse ADAM6gene expression and fertility.

Example 8 Characterization of ADAM6 Rescue Mice

Flow Cytometry. Three mice at age 25 weeks homozygous for human heavyand human κ light chain variable gene loci (H^(+/+)κ^(+/+)) and threemice at age 18-20 weeks homozygous for human heavy and human κ lightchain having the ectopic mouse genomic fragment encoding the mouseADAM6a and ADAM6b genes within both alleles of the human heavy chainlocus (H^(+/+)A6^(res)κ^(+/+)) were sacrificed for identification andanalysis of lymphocyte cell populations by FACs on the BD LSR II System(BD Bioscience). Lymphocytes were gated for specific cell lineages andanalyzed for progression through various stages of B cell development.Tissues collected from the animals included blood, spleen and bonemarrow. Blood was collected into BD microtainer tubes with EDTA (BDBiosciences). Bone marrow was collected from femurs by flushing withcomplete RPMI medium supplemented with fetal calf serum, sodiumpyruvate, HEPES, 2-mercaptoethanol, non-essential amino acids, andgentamycin. Red blood cells from blood, spleen and bone marrowpreparations were lysed with an ammonium chloride-based lysis buffer(e.g., ACK lysis buffer), followed by washing with complete RPMI medium.

For staining of cell populations, 1×10⁶ cells from the various tissuesources were incubated with anti-mouse CD16/CD32 (2.4G2, BD Biosciences)on ice for 10 minutes, followed by labeling with one or a combination ofthe following antibody cocktails for 30 minutes on ice.

Bone marrow: anti-mouse FITC-CD43 (1B11, BioLegend), PE-ckit (2B8,BioLegend), PeCy7-IgM (II/41, eBioscience), PerCP-Cy5.5-IgD (11-26c.2a,BioLegend), APC-eFluor780-B220 (RA3-6B2, eBioscience), A700-CD19 (1D3,BD Biosciences).

Peripheral blood and spleen: anti-mouse FITC-κ (187.1, BD Biosciences),PE-λ (RML-42, BioLegend), PeCy7-IgM (II/41, eBioscience),PerCP-Cy5.5-IgD (11-26c.2a, BioLegend), APC-CD3 (145-2C11, BD),A700-CD19 (1D3, BD), APC-eFluor780-B220 (RA3-6B2, eBioscience).Following incubation with the labeled antibodies, cells were washed andfixed in 2% formaldehyde. Data acquisition was performed on an LSRIIflow cytometer and analyzed with FlowJo (Treestar, Inc.). Results from arepresentative H^(+/+)κ^(+/+) and H^(+/+)A6^(res)κ^(+/+) mouse are shownin FIGS. 14-18.

The results demonstrate that B cells of H^(+/+)A6^(res)κ^(+/+) miceprogress through the stages of B cell development in a similar fashionto H^(+/+)κ^(+/+) mice in the bone marrow and peripheral compartments,and show normal patterns of maturation once they enter the periphery.H^(+/+)A6^(res)κ^(+/+) mice demonstrated an increased CD43^(int)CD19⁺cell population as compared to H^(+/+)κ^(+/+) mice (FIG. 16B). This mayindicate an accelerated IgM expression from the humanized heavy chainlocus containing an ectopic mouse genomic fragment comprising the mouseADAM6a and ADAM6b sequences in H^(+/+)A6^(res)κ^(+/+) mice. In theperiphery, B and T cell populations of H^(+/+)A6^(res)κ^(+/+) miceappear normal and similar to H^(+/+)κ^(+/+) mice.

Testis Morphology and Sperm Characterization.

To determine if infertility in mice having humanized immunoglobulinheavy chain variable loci is due to testis and/or sperm productiondefects, testis morphology and sperm content of the epididymis wasexamined.

Briefly, testes from two groups (n=5 per group; group 1: mice homozygousfor human heavy and κ light chain variable gene loci, H^(+/+)κ^(+/+);group 2: mice heterozygous for human heavy chain variable gene loci andhomozygous for κ light chain variable gene loci, H^(+/−)κ^(+/+)) weredissected with the epididymis intact and weighed. The specimens werethen fixed, embedded in paraffin, sectioned and stained with hematoxylinand eosin (HE) stain. Testis sections (2 testes per mouse, for a totalof 20) were examined for defects in morphology and evidence of spermproduction, while epididymis sections were examined for presence ofsperm.

In this experiment, no differences in testis weight or morphology wasobserved between H^(+/+)κ^(+/+) mice and H^(+/−)κ^(+/+) mice. Sperm wasobserved in both the testes and the epididymis of all genotypes. Theseresults establish that the absence of mouse ADAM6a and ADAM6b genes doesnot lead to detectable changes in testis morphology, and that sperm isproduced in mice in the presence and absence of these two genes. Defectsin fertility of male H^(+/+)κ^(+/+) mice are therefore not likely to bedue to low sperm production.

Sperm Motility and Migration.

Mice that lack other ADAM gene family members are infertile due todefects in sperm motility or migration. Sperm migration is defined asthe ability of sperm to pass from the uterus into the oviduct, and isnormally necessary for fertilization in mice. To determine if thedeletion of mouse ADAM6a and ADAM6b affects this process, spermmigration and motility was evaluated in H^(+/+)κ^(+/+) mice.

Briefly, sperm was obtained from testes of (1) mice heterozygous forhuman heavy chain variable gene loci and homozygous for human κ lightchain variable gene loci (H^(+/−)κ^(+/+)); (2) mice homozygous for humanheavy chain variable gene loci and homozygous for human κ light chainvariable gene loci (H^(+/+)κ^(+/+)); (3) mice homozygous for human heavychain variable gene loci and homozygous for wild-type κ light chain(H^(+/+)mκ); and, (4) wild-type C57 BL/6 mice (WT). No significantabnormalities were observed in sperm count or overall sperm motility byinspection. For all mice, cumulus dispersal was observed, indicatingthat each sperm sample was able to penetrate the cumulus cells and bindthe zona pellucida in vitro. These results establish that H^(+/+)κ^(+/+)mice have sperm that are capable of penetrating the cumulus and bindingthe zona pellucida.

Fertilization of mouse ova in vitro (IVF) was done using sperm from miceas described above. A slightly lower number of cleaved embryos wereobserved for H^(+/+)κ^(+/+) mice the day following IVF, as well as areduced number of sperm bound to the eggs. These results establish thatsperm from H^(+/+)κ^(+/+) mice, once exposed to an ovum, are capable ofpenetrating the cumulus and binding the zona pellucida.

In another experiment, the ability of sperm from H^(+/+)κ^(+/+) mice tomigrate from the uterus and through the oviduct was determined in asperm migration assay.

Briefly, a first group of super-ovulated female mice (n=5) were set upwith H^(+/+)κ^(+/+) males (n=5) and a second group of super-ovulatedfemale mice (n=5) were set up with H^(+/−)κ^(+/+) males (n=5). Themating pairs were observed for copulation, and five to six hourspost-copulation the uterus and attached oviduct from all females wereremoved and flushed for analysis. Flush solutions were checked for eggsto verify ovulation and obtain a sperm count. Sperm migration wasevaluated in two different ways. First, both oviducts were removed fromthe uterus, flushed with saline, and any sperm identified were counted.The presence of eggs was also noted as evidence of ovulation. Second,oviducts were left attached to the uterus and both tissues were fixed,embedded in paraffin, sectioned and stained (as described above).Sections were examined for presence of sperm, in both the uterus and inboth oviducts.

For the females mated with the five H^(+/+)κ^(+/+) males, very littlesperm was found in the flush solution from the oviduct. Flush solutionsfrom oviducts of the females mated with the H^(+/−)κ^(+/+) malesexhibited a sperm level about 25- to 30-fold higher (avg, n=10 oviducts)than present in flush solutions from the oviducts of the females matedwith the H^(+/+)κ^(+/+) males. A representative breeding comparison ofH^(+/+)κ^(+/+) and H^(+/+)A6^(res)κ^(+/+) mice is shown in Table 9.

Histological sections of uterus and oviduct were prepared. The sectionswere examined for sperm presence in the uterus and the oviduct (thecolliculus tubarius). Inspection of histological sections of oviduct anduterus revealed that for female mice mated with H^(+/+)κ^(+/+) mice,sperm was found in the uterus but not in the oviduct. Further, sectionsfrom females mated with H^(+/+)κ^(+/+) mice revealed that sperm was notfound at the uterotubal junction (UTJ). In sections from females matedwith H^(+/−)κ^(+/+) mice, sperm was identified in the UTJ and in theoviduct.

These results establish that mice lacking ADAM6a and ADAM6b genes makesperm that exhibit an in vivo migration defect. In all cases, sperm wasobserved within the uterus, indicating that copulation and sperm releaseapparently occur as normal, but little to no sperm was observed withinthe oviducts after copulation as measured either by sperm count orhistological observation. These results establish that mice lackingADAM6a and ADAM6b genes produce sperm that exhibit an inability tomigrate from the uterus to the oviduct. This defect apparently leads toinfertility because sperm are unable to cross the uterine-tubulejunction into the oviduct, where eggs are fertilized. Taken together,all of these results converge to the support the hypothesis that mouseADAM6 genes help direct sperm with normal motility to migrate out of theuterus, through the uterotubal junction and the oviduct, and thusapproach an egg to achieve the fertilization event. The mechanism bywhich ADAM6 achieves this may be directed by one or both of the ADAM6proteins, or through coordinate expression with other proteins, e.g.,other ADAM proteins, in the sperm cell, as described below.

TABLE 9 Breeding Male Animals Duration of Genotype (Male/Female)Breeding Litters Offspring H^(+/+)κ^(+/+) 6/6 6 months 2 25H^(+/+)A6^(res)κ^(+/+) 4/8 4 months 4 198

ADAM Gene Family Expression.

A complex of ADAM proteins are known to be present as a complex on thesurface of maturing sperm. Mice lacking other ADAM gene family memberslose this complex as sperm mature, and exhibit a reduction of multipleADAM proteins in mature sperm. To determine if a lack of ADAM6a andADAM6b genes affects other ADAM proteins in a similar manner, Westernblots of protein extracts from testis (immature sperm) and epididymis(maturing sperm) were analyzed to determine the expression levels ofother ADAM gene family members.

In this experiment, protein extracts were analyzed from groups (n=4 pergroup) of H^(+/+)κ^(+/+) and H^(+/−)κ^(+/+) mice. The results showedthat expression of ADAM2 and ADAM3 were not affected in testis extracts.However, both ADAM2 and ADAM3 were dramatically reduced in epididymisextracts. This demonstrates that the absence of ADAM6a and ADAM6b insperm of H^(+/+)κ^(+/+) mice may have a direct affect on the expressionand perhaps function of other ADAM proteins as sperm matures (e.g.,ADAM2 and ADAM3). This suggests that ADAM6a and ADAM6b are part of anADAM protein complex on the surface of sperm, which might be criticalfor proper sperm migration.

Example 9 Human Heavy Chain Variable Gene Utilization in ADAM6 RescueMice

Selected human heavy chain variable gene usage was determined for micehomozygous for human heavy and κ light chain variable gene loci eitherlacking mouse ADAM6a and ADAM6b genes (H^(+/+)κ^(+/+)) or containing anectopic genomic fragment encoding for mouse ADAM6a and ADAM6b genes(H^(+/+)A6^(res)κ^(+/+)) by a quantitative PCR assay using TAQMAN™probes (as described above).

Briefly, CD19⁺ B cells were purified from the spleens of H^(+/+)κ^(+/+)and H^(+/+)A6^(res)κ^(+/+) mice using mouse CD19 Microbeads (MiltenyiBiotec) and total RNA was purified using the RNEASY™ Mini kit (Qiagen).Genomic RNA was removed using an RNase-free DNase on-column treatment(Qiagen). About 200 ng mRNA was reverse-transcribed into cDNA using theFirst Stand cDNA Synthesis kit (Invitrogen) and then amplified with theTAQMAN™ Universal PCR Master Mix (Applied Biosystems) using the ABI 7900Sequence Detection System (Applied Biosystems). Relative expression ofeach gene was normalized to the expression of mouse κ light chainconstant region (mCκ). Table 10 sets forth the sense/antisense/TAQMAN™MGB probe combinations used in this experiment.

TABLE 10 Human SEQ V_(H) Sequence (5′-3′) ID NO: V_(H)6-1Sense: CAGGTACAGCTGCAGCAGTCA 6 Anti-sense: GGAGATGGCACAGGTGAGTGA 7Probe: TCCAGGACTGGTGAAGC 8 V_(H)1-2 Sense: TAGTCCCAGTGATGAGAAAGAGAT 9Anti-sense: GAGAACACAGAAGTGGATGAGATC 10 Probe: TGAGTCCAGTCCAGGGA 11V_(H)3-23 Sense: AAAAATTGAGTGTGAATGGATAAGAGTG 12Anti-sense: AACCCTGGTCAGAAACTGCCA 13 Probe: AGAGAAACAGTGGATACGT 14V_(H)1-69 Sense: AACTACGCACAGAAGTTCCAGG 15Anti-sense: GCTCGTGGATTTGTCCGC 16 Probe: CAGAGTCACGATTACC 17 mC_(κ)Sense: TGAGCAGCACCCTCACGTT 18 Anti-sense: GTGGCCTCACAGGTATAGCTGTT 19Probe: ACCAAGGACGAGTATGAA 20

In this experiment, expression of all four human V_(H) genes wasobserved in the samples analyzed. Further, the expression levels werecomparable between H^(+/+)κ^(+/+) and H^(+/+)A6^(res)κ^(+/+) mice. Theseresults demonstrate that human V_(H) genes that were both distal to themodification site (V_(H)3-23 and V_(H)1-69) and proximal to themodification site (V_(H)1-2 and V_(H)6-1) were all able to recombine toform a functionally expressed human heavy chain. These resultsdemonstrate that the ectopic genomic fragment comprising mouse ADAM6aand ADAM6b sequences inserted into a human heavy chain genomic sequencedid not affect V(D)J recombination of human heavy chain gene segmentswithin the locus, and these mice are able to recombine human heavy chaingene segments in normal fashion to produce functional heavy chainimmunoglobulin proteins.

Example 10 Humoral Immune Response in ADAM6 Rescue Mice

The humoral immune response was determined for mice homozygous for humanheavy and κ light chain variable gene loci either lacking mouse ADAM6aand ADAM6b genes (H^(+/+)κ^(+/+)) or containing an ectopic genomicfragment encoding for mouse ADAM6a and ADAM6b genes(H^(+/+)A6^(res)κ^(+/+)) by a multi-antigen immunization scheme followedby antibody isolation and characterization. Results were compared fordetermination of any effect on V(D)J recombination involving the humanimmunoglobulin gene segments, assessment of serum titer progression,production of antibodies by hybridomas and affinity for antigen.

Immunization Protocol.

A human cell surface receptor (Antigen A), a human antibody specific fora human receptor tyrosine-protein kinase (Antigen B), a secreted humanprotein that functions in regulation of the TGF-6 signaling pathway(Antigen C), and a human receptor tyrosine kinase (Antigen D) wereemployed for comparative immunizations in groups of mice. Serum wascollected from groups of mice prior to immunization with the aboveantigens. Each antigen (2.3 μg each) was administered in an initialpriming immunization mixed with 10 μg of CpG oligonucleotide as adjuvant(Invivogen). The immunogen was administered via footpad (f.p.) in avolume of 25 μl per mouse. Subsequently, mice were boosted via f.p. with2.3 μg of antigen along with 10 μg CpG and 25 μg Adju-Phos (Brenntag) asadjuvants on days 3, 6, 11, 13, 17, and 20 for a total of six boosts.Mice were bled on days 15 and 22 after the fourth and sixth boosts,respectively, and antisera were assayed for antibody titer to eachspecific antigen.

Antibody titers were determined in sera of immunized mice using an ELISAassay. Ninety six-well microliter plates (Thermo Scientific) were coatedwith the respective antigen (2 μg/ml) in phosphate-buffered saline (PBS,Irvine Scientific) overnight at 4° C. The following day, plates werewashed with phosphate-buffered saline containing 0.05% Tween 20 (PBS-T,Sigma-Aldrich) four times using a plate washer (Molecular Devices).Plates were then blocked with 250 μl of 0.5% bovine serum albumin (BSA,Sigma-Aldrich) in PBS and incubated for one hour at room temperature.The plates were then washed four times with PBS-T. Sera from immunizedmice and pre-immune sera were serially diluted three-fold in 0.5%BSA-PBS starting at 1:300 or 1:1000 and added to the blocked plates induplicate and incubated for one hour at room temperature. The last twowells were left blank to be used as secondary antibody control. Theplates were again washed four times with PBS-T in a plate washer. A1:5000/1:10,000 dilution of goat anti-mouse IgG-Fc-Horse RadishPeroxidase (HRP, Jackson Immunoresearch) or goat anti-mouseIgG-kappa-HRP (Southern Biotech) conjugated secondary antibody was addedto the plates and incubated for one hour at room temperature. Plateswere again washed eight times with PBS-T and developed using TMB/H₂O₂ assubstrate. The substrate was incubated for twenty minutes and thereaction stopped with 2 N H₂SO₄(VWR) or 1 N H₃PO₄ (JT Baker). Plateswere read on a spectrophotometer (Victor, Perkin Elmer) at 450 nm.Antibody titers were calculated using Graphpad PRISM software.

Serum titer was calculated as serum dilution within experimentaltitration range at the signal of antigen binding equivalent to two timesabove background. Results for the humoral immune response are shown inFIG. 19 (Antigen A), FIG. 20 (Antigen B), FIG. 21 (Antigen C), and FIG.22 (Antigen D). Antigen positive score of hybridomas made using twospleens isolated from mice from each group of selected immunizations isshown in Table 11 (Antigen score is equal to 2×/background).

As shown in this Example, antibody titers generated in Adam6 rescue mice(H^(+/+)A6^(res)κ^(+/+)) were comparable to those generated in micelacking ADAM6a and ADAM6b and having humanized heavy chain(H^(+/+)κ^(+/+)). Further, spleens from H^(+/+)A6^(res)κ^(+/+) miceyielded antigen-positive hybridomas for all antigens tested, includingantibodies of high affinity, at levels comparable to H^(+/+)κ^(+/+)mice. Thus, no impairment of V(D)J recombination of human immunoglobulingene segments in Adam6 rescue mice is believed to exist given theproduction of antibodies with high affinity containing humanimmunoglobulin genes.

TABLE 11 Antigen Mouse Strain Antigen Score A H^(+/+)A6^(res)κ^(+/+) 76A H^(+/+)A6^(res)κ^(+/+) 32 B H^(+/+)κ^(+/+) 4 B H^(+/+)κ^(+/+) 12 BH^(+/+)A6^(res)κ^(+/+) 41 B H^(+/+)A6^(res)κ^(+/+) 95

Example 11 Antigen Binding Affinity Determination

Binding affinities of antibodies showing specific binding to Antigen Bwere screened using a real-time surface plasmon resonance biosensor(BIAcore 2000). Conditioned media from hybridomas isolated from twostrains of mice immunized with Antigen B (H^(+/+)κ^(+/+) andH^(+/+)A6^(res)κ^(+/+)) were used during BIAcore screening. BIAcoresensor surface was first derivatized with polyclonal rabbit anti-mouseantibody (GE) to capture anti-Antigen B antibodies from conditionedmedia. During the entire screening method, HBST (0.01 M HEPES pH 7.4,0.15M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) was used as therunning buffer. Fab fragment of Antigen B was injected over theanti-Antigen B antibody captured surface at a flow rate of 50 μl/minuteat 100 nM concentration. Antibody-antigen association was monitored forthree minutes while the dissociation of antigen from the capturedantibody was monitored for five minutes in HBST running buffer. Theexperiment was performed at 25° C. Kinetic association (ka) anddissociation (kd) rate constants were determined by processing andfitting the data to a 1:1 binding model using Scrubber 2.0 curve fittingsoftware. Binding dissociation equilibrium constants (K_(D)) anddissociative half-lives (T_(1/2)) were calculated from the kinetic rateconstants as: K_(D) (M)=kd I ka; and T % (min)=(ln 2/(60*kd). Resultsfor selected anti-Antigen B antibodies are shown in Table 12.

TABLE 12 Antibody Mouse strain K_(D) (M) T_(1/2) (min) 5D6H^(+/+)κ^(+/+) 1.62E−08 3 8G10 H^(+/+)κ^(+/+) 1.20E−08 5 10F10H^(+/+)κ^(+/+) 1.09E−08 3 1F5 H^(+/+)κ^(+/+) 1.00E−07 0.3 10G8H^(+/+)κ^(+/+) 1.47E−07 0.3 1B11 H^(+/+)A6^(res)κ^(+/+) 1.98E−08 6 2D9H^(+/+)A6^(res)κ^(+/+) 9.40E−10 51 4D11 H^(+/+)A6^(res)κ^(+/+) 5.60E−080.8 6C5 H^(+/+)A6^(res)κ^(+/+) 1.10E−09 188 6F4 H^(+/+)A6^(res)κ^(+/+)1.35E−08 3 7C4 H^(+/+)A6^(res)κ^(+/+) 2.00E−06 0.05 8G12H^(+/+)A6^(res)κ^(+/+) 2.31E−09 19 9B12 H^(+/+)A6^(res)κ^(+/+) 3.47E−0913 10B4 H^(+/+)A6^(res)κ^(+/+) 3.60E−09 23 11E7 H^(+/+)A6^(res)κ^(+/+)3.06E−08 2 11E12 H^(+/+)A6^(res)κ^(+/+) 2.70E−07 0.1 1E4H^(+/+)A6^(res)κ^(+/+) 7.00E−10 58 4D2 H^(+/+)A6^(res)κ^(+/+) 5.80E−10150 5H6 H^(+/+)A6^(res)κ^(+/+) 2.60E−09 3 5H10 H^(+/+)A6^(res)κ^(+/+)6.00E−09 70 9A9 H^(+/+)A6^(res)κ^(+/+) 3.80E−09 12 11C11H^(+/+)A6^(res)κ^(+/+) 1.55E−09 38 12C10 H^(+/+)A6^(res)κ^(+/+) 5.90E−0916 12G7 H^(+/+)A6^(res)κ^(+/+) 9.00E−08 7 12G9 H^(+/+)A6^(res)κ^(+/+)3.32E−09 12

In a similar experiment, kinetics of different monoclonal antibodiespresent in hybridoma-conditioned media binding to Antigen A wasdetermined using a real-time surface plasmon resonance biosensor(BIAcore 4000) assay. All hybridoma clones used in this assay wereproduced in H^(+/+)A6^(res)κ^(+/+) mice.

Briefly, to capture the Antigen A-specific antibodies, polyclonal rabbitanti-mouse antibody (GE Catalog# BR-1008-38) was first immobilized onthe sensor chip. BIAcore screening was performed in two differentbuffers—PBSP, pH7.2 and PBSP, pH6.0. Both the buffers were supplementedwith 0.1 mg/ml BSA. Following the capture of anti-Antigen A antibodiesfrom the conditioned media, 1 μM of Antigen A monomer (prepared inrespective running buffer) was injected over the captured antibodysurface for 1.5 minutes at 30 μl/minute and the dissociation of boundAntigen A monomer was monitored for 1.5 minutes in the respectiverunning buffer at 25° C. Kinetic association (ka) and dissociation (kd)rate constants were determined by processing and fitting the data to a1:1 binding model using Scrubber 2.0 curve fitting software. Bindingdissociation equilibrium constants (K_(D)) and dissociative half-lives(T_(x)) were calculated from the kinetic rate constants as: K_(D)(M)=kd/ka; and T_(y), (min)=(ln 2/(60*kd). Table 13 sets forth thebinding kinetics parameters for selected anti-Antigen A antibody bindingto Antigen A monomer at pH7.2 and pH6.0. NB: no binding detected undercurrent experimental conditions.

TABLE 13 pH 7.2 pH 6.0 Antibody K_(D) (M) T_(1/2) (min) K_(D) (M)T_(1/2) (min) 1D7 3.89E−10 25 9.45E−10 17 2B4 NB NB NB NB 2B7 3.90E−091.2 2.98E−09 2 2F7 2.36E−10 144 2.06E−11 1882 3A7 NB NB 6.42E−10 17 3F6NB NB NB NB 4A6 1.91E−09 2 2.12E−09 2 4C4 NB NB NB NB 4E12 2.69E−10 162.03E−10 18 5C11 1.68E−09 3 2.31E−09 3 5D10 NB NB 4.56E−09 2 5E7 NB NBNB NB 5F10 NB NB NB NB 5F11 8.18E−10 8 6.79E−10 7 5G4 3.55E−10 157.42E−11 53 5G9 6.39E−10 15 4.31 E−10 21 5H8 4.73E−10 15 NB NB 6D2 NB NBNB NB 6D3 2.88E−10 14 8.82E−11 39 6E4 NB NB 2.67E−09 4 6E6 1.37E−09 101.30E−09 14 6H6 NB NB NB NB 7A12 NB NB NB NB 7C3 NB NB NB NB 7E84.38E−10 22 2.63E−10 34 7F10 NB NB NB NB 7G9 NB NB NB NB 8B8 NB NB NB NB8B11 NB NB NB NB 8C3 NB NB NB NB 8E9 NB NB NB NB 8G3 NB NB NB NB 8H3 NBNB NB NB 8H4 3.70E−07 0.1 NB NB 8H8 NB NB NB NB 1A8 2.30E−09 4 7.40E−106 1B6 NB NB NB NB 1C6 NB NB NB NB 1C12 NB NB NB NB 1D2 NB NB NB NB 1E21.17E−09 42 3.08E−09 29 1E3 5.05E−10 89 8.10E−10 57 1E6 1.97E−08 31.84E−08 3 1E9 1.14E−09 30 1.14E−09 25 1H6 2.93E−09 14 9.87E−10 25 2H92.30E−08 2 1.91E−08 2 3A2 1.15E−10 44 1.25E−10 33 3A4 1.70E−10 311.44E−10 30 3D11 NB NB 1.58E−08 1 3H10 2.82E−09 20 2.59E−09 15 4B67.79E−10 6 6.36E−10 7 4H6 9.18E−11 62 1.20E−10 43 5A2 NB NB 7.04E−10 125C5 8.71E−11 49 7.02E−11 48 5F6 6.16E−11 114 5.46E−11 121

As shown above, high affinity antibodies were obtained from bothH^(+/+)A6^(res)κ^(+/+) and H^(+/+)κ^(+/+) mice in a comparable manner.Among the twenty-five antibodies represented in Table 12, twentyproduced in H^(+/+)A6^(res)κ^(+/+) mice demonstrated an affinity rangeof 0.5 nM to 1 μM, while the five generated in H^(+/+)κ^(+/+) micedemonstrated an affinity range of 10 nM to 150 nM. Further, thefifty-five antibodies shown in Table 13 demonstrated an affinity rangeof 20 pM to 350 nM for binding to Antigen A monomer.

As demonstrated in this Example, the reinsertion of mouse Adam6 genesinto a humanized immunoglobulin heavy chain locus does not impair theability of the mouse to mount a robust immunize response to multipleantigens characterized by repertoires of human antibodies having diverseaffinities in the subnanomolar range, which are derived from human genesegments rearranged from a engineered germline.

1.-27. (canceled)
 28. A genetically modified mouse whose genome: (i)lacks a functional endogenous ADAM6 locus; and (ii) includes anintegrated nucleic acid sequence encoding a mouse ADAM6 protein.
 29. Thegenetically modified mouse of claim 28, wherein the integrated nucleicacid sequence encoding a mouse ADAM6 protein is not found between thetwo mouse immunoglobulin variable region sequences in which it is foundin a wild-type mouse.
 30. A genetically modified mouse whose genome: (i)lacks a functional endogenous ADAM6 locus; and (ii) includes anextrachromosomal nucleic acid sequence encoding a mouse ADAM6 protein.31. The genetically modified mouse of claim 28 or claim 30, whose genomecomprises a deletion of an endogenous immunoglobulin variable regionsequence.
 32. The genetically modified mouse of claim 31, wherein thedeletion comprises a deletion of a mouse V_(H) gene segment, a deletionof a mouse D_(H) gene segment, a deletion of a mouse J_(H) gene segment,and combinations thereof.
 33. A method of increasing mouse fertility by:integrating into a mouse cell a nucleic acid sequence encoding a mouseADAM6 protein.
 34. The method of claim 33, wherein the mouse cellcomprises a deletion of an endogenous heavy chain variable region. 35.The method of claim 33, wherein the mouse cell comprises a replacementof an endogenous heavy chain variable region.
 36. The method of claim33, wherein the mouse comprises human immunoglobulin V_(H) segments. 37.The method of claim 36, wherein the mouse comprises a humanizedimmunoglobulin heavy chain locus in that the human immunoglobulin V_(H)segments are at the endogenous mouse immunoglobulin heavy chain locus.38. The method of claim 37, wherein the integrated nucleic acid sequenceencoding a mouse ADAM6 protein is within the humanized immunoglobulinheavy chain locus.
 39. The method of claim 38, wherein the humanizedimmunoglobulin heavy chain locus comprises replacement of mouse germlinevariable region segments with the human immunoglobulin V_(H) segments.40. A genetically modified mouse produced by the method of claim
 33. 41.A genetically modified mouse whose genome: (i) lacks a functionalendogenous ADAM6 locus; and (ii) includes an integrated orextrachromosomal nucleic acid sequence encoding a mouse ADAM6 protein.42. The genetically modified mouse of claim 41, wherein the mouse has agenome that comprises an endogenous ADAM6 locus or fragment thereof,which endogenous ADAM6 locus or fragment thereof does not express ADAM6.43. The genetically modified mouse of claim 41, wherein the mouse has agenome that comprises an endogenous ADAM6 locus or fragment thereof,which endogenous ADAM6 locus or fragment thereof expresses ADAM6 at alevel that is insufficient to support essentially normal fertility of amale mouse.
 44. The mouse of any one of claim 42 or 43, wherein theendogenous ADAM6 locus or fragment thereof has a modification in astructural gene selected from the group consisting of ADAM6a, ADAM6b,and combinations thereof.
 45. The mouse of any one of claim 42 or 43,wherein the endogenous ADAM6 locus or fragment thereof has amodification in a regulatory region.
 46. The modified mouse of claim 45,wherein the regulatory region is selected from the group consisting of aregion 5′ to the ADAM6a gene, a region 3′ of the ADAM6a coding region, aregion 3′ of the ADAM6b coding region, and combinations thereof.
 47. Thegenetically modified mouse of claim 41, wherein the integrated orextrachromosomal nucleic acid sequence is not found between the twomouse immunoglobulin variable region sequences in which it is found in awild-type mouse.
 48. A genetically modified mouse whose genome comprisesa replacement of one or more endogenous V_(H) gene segments with humanV_(H) gene segments, wherein the replacement removes an endogenous ADAM6sequence, which mouse is engineered to have a mouse ADAM6 sequencelocated within the sequence that contains the human V_(H) gene segments.49. The genetically modified mouse of claim 48, wherein the mouse ADAM6sequence is placed between two human V_(H) gene segments, where thehuman V_(H) gene segments are operably linked to D_(H) and J_(H) genesegments.
 50. The genetically modified mouse of claim 49, wherein theD_(H) segments are human.
 51. The genetically modified mouse of claim49, wherein the D_(H) segments are mouse.
 52. The genetically modifiedmouse of claim 49, wherein the J_(H) segments are human.
 53. Thegenetically modified mouse of claim 49, wherein the J_(H) segments aremouse.
 54. The genetically modified mouse of 49, wherein the human V_(H)gene segments are operably linked to D_(H) and J_(H) gene segments andto an immunoglobulin constant region.
 55. The genetically modified mouseof claim 54, wherein the constant region is human.
 56. The geneticallymodified mouse of claim 54, wherein the constant region is mouse.
 57. Amouse immunoglobulin heavy chain locus that lacks an endogenous mouseADAM6 sequence, which locus is humanized in that it contains a sequencethat includes human V_(H) gene segments, wherein the mouseimmunoglobulin heavy chain locus includes a mouse ADAM6 gene within thesequence that includes human V_(H) gene segments.
 58. The mouseimmunoglobulin heavy chain locus of claim 57, wherein the sequence thatincludes human V_(H) gene segments comprises a replacement one or moreendogenous V_(H) gene segments with human V_(H) gene segments.
 59. Themouse immunoglobulin heavy chain locus of claim 58, wherein thereplacement removes the endogenous ADAM6 sequence.
 60. The mouseimmunoglobulin heavy chain locus of claim 58, wherein the mouse ADAM6sequence is placed between two human V_(H) gene segments.
 61. The mouseimmunoglobulin heavy chain locus of claim 58, wherein the humanizedimmunoglobulin heavy chain locus comprises human V_(H) gene segmentsoperably linked to human or mouse D_(H) and J_(H) gene segments, and toa human or mouse immunoglobulin constant region.
 62. A geneticallymodified mouse whose genome comprises the mouse immunoglobulin heavychain locus of claim
 57. 63. A mouse whose genome includes animmunoglobulin heavy chain locus that lacks any mouse ADAM6 sequence,which locus is humanized in that it contains a sequence that includeshuman V_(H) gene segments, wherein the mouse genome includes anintegrated mouse ADAM6 gene.
 64. A mouse immunoglobulin heavy chainlocus that comprises a nonfunctional endogenous mouse ADAM6 sequence orregulatory region, and the locus comprises an ectopic nucleotidesequence that encodes a mouse ADAM6 protein or a homolog or ortholog ora functional fragment thereof.
 65. In a genetically modified mousecomprising a humanized immunoglobulin heavy chain locus that compriseshuman V_(H) gene segments operably linked to a human or mouseimmunoglobulin constant region, the improvement comprising: a geneticmodification that introduces into the mouse a functional gene selectedfrom the group consisting of mouse ADAM6a, mouse ADAM6b, andcombinations thereof.
 66. The improvement of claim 65, wherein thefunctional gene is introduced in a transgene that randomly integratesinto the mouse genome.
 67. The improvement of claim 56, wherein thefunctional gene is maintained episomally.
 68. In a genetically modifiedmouse in which endogenous heavy chain immunoglobulin gene segmentsselected from the group consisting of V gene segments and D genesegments are deleted or replaced, the improvement comprising: a geneticmodification that introduces into the mouse a functional gene selectedfrom the group consisting of mouse ADAM6a, mouse ADAM6b, andcombinations thereof.
 69. The improvement of claim 68, wherein thefunctional gene is introduced in a transgene that randomly integratesinto the mouse genome.
 70. The improvement of claim 68, wherein thefunctional gene is maintained episomally.
 71. In a genetically modifiedmouse in which an endogenous immunoglobulin variable gene segment andADAM6 nucleotide sequence has been replaced with a sequence comprising ahuman immunoglobulin variable region gene segment to form a firstchimeric locus, the improvement comprising: insertion of a mouseADAM6-encoding sequence into the sequence comprising the humanimmunoglobulin variable region gene segment to form a second chimericlocus.
 72. The improvement of claim 71, wherein the second chimericlocus comprises a human immunoglobulin heavy chain variable (V_(H))segment.
 73. The improvement of claim 71 or claim 72, wherein the secondchimeric locus comprises a human immunoglobulin light chain variable(V_(L)) segment.
 74. The improvement of claim 71, wherein the secondchimeric locus comprises a human V_(H) segment or a human V_(L) segmentoperably linked to a human D_(H) segment and a human J_(H) segment. 75.The improvement of claim 71, wherein the second chimeric locus isoperably linked to a third chimeric locus that comprises a human C_(H)1sequence, or a human C_(H)1 and human hinge sequence, fused with a mouseC_(H)2 and C_(H)3 sequence.
 76. The improvement of claim 71, wherein theimprovement comprises increased fertility of the genetically modifiedmouse.
 77. A genetically modified mouse whose genome comprises a humanimmunoglobulin heavy chain variable region gene segment at an endogenousmouse immunoglobulin heavy chain variable region gene locus andcomprises a nucleotide sequence that expresses mouse ADAM6, whichnucleotide sequence is extrachromosomal or is integrated in the genomeof the mouse.
 78. A genetically engineered mouse from which heavy chainV gene segments are deleted or replaced, or from which heavy chain Dgene segments are deleted or replaced, which mouse has been modified toinclude a nucleotide sequence that encodes a mouse ADAM6 proteinselected from the group consisting of mouse ADAM6a, mouse ADAM6b, andcombinations thereof.
 79. The genetically engineered mouse of claim 78,wherein the included nucleotide sequence is or comprises a transgenethat randomly integrates into the mouse genome.
 80. The geneticallyengineered mouse of claim 78, wherein the included nucleotide sequenceis maintained episomally.
 81. A genetically engineered mouse whosegenome comprises: a) endogenous immunoglobulin loci that are disabled;b) a human transgene that comprises variable and constant immunoglobulingene segments; and further comprise a nucleic acid sequence encoding amouse ADAM6, which nucleic acid sequence is ectopic in that it isextrachromosomal or is integrated in a transcriptionally-permissivelocus that is not between the two mouse immunoglobulin variable regionsequences in which it is found in a wild-type mouse.
 82. A geneticallyengineered mouse whose genome comprises hybrid immunoglobulin loci thatmake light and heavy chains that have a human variable region and amouse constant region or that have a human variable regions and partlyhuman/partly mouse constant regions, which genetically engineered mousefurther comprises an extrachromosomal or integrated nucleic acidencoding a mouse ADAM6.
 83. The genetically engineered mouse of claim 81or claim 82, wherein the human variable gene segments comprise V_(H)1-2and V_(H)6-1.
 84. The genetically engineered mouse of claim 83, whereinthe nucleic acid sequence encoding the mouse ADAM6 is placed between thehuman V_(H)1-2 and the human V_(H)6-1 gene segments.
 85. The geneticallyengineered mouse of claim 84, wherein the nucleic acid sequence encodingthe mouse ADAM6 has a direction of transcription opposite of that of thesurrounding human variable gene segments.
 86. The genetically engineeredmouse of claim 81 or 82, wherein the nucleic acid sequence encoding amouse ADAM6 is positioned at a transcriptionally-permissive locus in themouse genome.
 87. A genetically engineered mouse whose genome comprisesa disruption of an endogenous mouse ADAM6 locus and further comprises anextrachromosomal nucleotide sequence encoding a mouse ADAM6 protein. 88.A genetically engineered mouse whose genome comprises a deletion of anendogenous mouse ADAM6 locus and further comprises an integratednucleotide sequence encoding a mouse ADAM6 protein.
 89. The geneticallyengineered mouse of claim 88, wherein the nucleotide sequence encoding amouse ADAM6 protein is integrated at a transcriptionally-permissivelocus in the mouse genome.
 90. A genetically engineered mouse whosegenome comprises a modification of an endogenous heavy chainimmunoglobulin locus that compromises endogenous ADAM6 function whichgenetically engineered mouse further comprises an extrachromosomal orintegrated nucleic acid encoding a mouse ADAM6.
 91. A geneticallyengineered mouse whose genome comprises: a) a heavy chain immunoglobulinlocus that is modified to be deleted in whole or in part, humanized inwhole or in part, or replaced in whole or in part; and b) a nucleotidesequence encoding a mouse ADAM6 protein that is ectopic in that it isextrachromosomal or is integrated in a transcriptionally-permissivelocus that is not between the two mouse immunoglobulin variable regionsequences in which it is found in a wild-type mouse.
 92. A nucleic acidconstruct comprising mouse ADAM6a and ADAM6b genes and furthercomprising sites engineered onto the 5′ and 3′ ends of the construct forintegration into a humanized immunoglobulin heavy chain locus.
 93. Agenetically engineered mouse whose genome comprises mouse ADAM6a andADAM6b genes integrated within a humanized immunoglobulin chain locus.94. A method of increasing fertility in a mouse, the method comprising:integrating into the genome of a mouse ES cell a targeting vectorcomprising mouse ADAM6a and ADAM6b genes, and introducing the ES cellinto an 8-cell-stage mouse embryo, so that a mouse that expressesfunctional mouse ADAM6 protein develops.
 95. A method of making agenetically engineered mouse, the method comprising steps of: gestatingin a surrogate mother a mouse embryo comprising an integrated mouseADAM6 gene.
 96. The method of claim 95, wherein the step of gestatingcomprises: integrating into the mouse embryo a nucleic acid sequenceencoding a mouse ADAM6 protein.
 97. The method of claim 96, wherein thestep of integrating into the mouse embryo comprises: integrating into adonor mouse ES cell or a mouse embryo a nucleic acid sequence encoding amouse ADAM6 protein; and introducing the donor mouse ES cell into theembryo.
 98. The method of claim 95, wherein the mouse into which themouse ADAM6 gene is integrated lacks an endogenous ADAM6 gene at anendogenous immunoglobulin heavy chain locus.
 99. A method ofmanufacturing a nucleic acid construct comprising mouse ADAM6a andADAM6b genes by positioning the genes between 5′ and 3′ sequences thattarget the mouse ADAM6a and ADAM6b genes for integration into one ormore loci in the mouse genome.
 100. The method of claim 99, wherein thesequences target the that target mouse ADAM6a and ADAM6b genes forintegration into an immunoglobulin heavy chain locus.
 101. The method ofclaim 99, wherein the sequences target the that target mouse ADAM6a andADAM6b genes for integration into a humanized immunoglobulin heavy chainlocus.
 102. A method of manufacturing a nucleic acid constructcomprising mouse ADAM6a and ADAM6b genes by positioning the genesbetween 5′ and 3′ sequences that do not include a complete mouseimmunoglobulin heavy chain locus and that direct random integration ofthe mouse ADAM6a and ADAM6b genes into the mouse genome.
 103. Agenetically engineered mouse cell comprising an integrated mouse ADAM6gene, which ADAM6 gene was integrated separate from the intact mouseimmunoglobulin heavy chain locus from which it originated.
 104. Thegenetically engineered mouse cell of claim 103, comprising integratedADAM6a and ADAM6b genes.
 105. A mouse B cell whose genome comprises: arearranged human immunoglobulin variable region gene sequence; and anintegrated mouse ADAM6 gene.
 106. The mouse B cell of claim 105, whereinthe rearranged human immunoglobulin variable gene sequence is selectedfrom the group consisting of a heavy chain variable gene sequence and alight chain variable gene sequence.
 107. The mouse B cell of claim 105or 106, wherein the rearranged human immunoglobulin variable region isoperably linked with a mouse constant region gene sequence.
 108. Themouse of any one of claims 28, 30, 40, 41, 48, 62, 63, 65, 68, 71, 77,78, 81, 82, 87, 88, 90, 91, 93 and 103, which mouse has B cells thatcomprise a rearranged human immunoglobulin variable region genesequence.
 109. The mouse of claim 108, wherein the rearranged humanimmunoglobulin variable gene sequence is selected from the groupconsisting of a heavy chain variable gene sequence and a light chainvariable gene sequence.
 110. The mouse of claim 109, wherein therearranged human immunoglobulin variable region is operably linked witha mouse constant region gene sequence.